LOW-MOLECULAR-WEIGHT CHROMIUM-BINDING SUBSTANCE:
ADVANCED STUDIES FROM AVES TO HUMAN
by
YUAN CHEN
A DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
in the Department of Chemistry
in the Graduate School of
The University of Alabama
TUSCALOOSA, ALABAMA
2009
Copyright Yuan Chen 2009
ALL RIGHTS RESERVED
ABSTRACT
Chromium has been observed to play a role in maintaining proper carbohydrate and lipid
metabolism of mammals. One of the potentially biological active forms of chromium in vitro is
low-molecular-weight chromium-binding substance (LMWCr), which has been proposed to
amplify the insulin cascade by binding with insulin receptor.
LWMCr is a small bio-molecule (<1500 Da) containing a carboxylate-rich polypeptide
with four bound chromic ions. LMWCr’s (according to its amino acid composition and mass
spectrum) were successfully isolated from chicken and alligator livers, as well as from human
urine, using a modified method. The extreme hydrophilicity of the peptide and the tightly bound
Cr(III) are two major hurdles to produce a stable end-product for mass spectrometry (MS) or
high-performance liquid chromatography (HPLC) analysis. Treating bovine LMWCr with
trifluoroacetic acid and application to a graphite powder micro-column was used to generate a
heptapeptide fragment, and the peptide sequence was analyzed by mass spectrometry (MS) and
tandem MS (MS/MS). Two candidate sequences, EEEEGDD and EEEGEDD, were identified;
the mass spectrum of the former sequence is more similar to that of the LMWCr fragment.
Langmuir isotherm and Hill plots were used to analyze the binding constants of chromic
ions to synthetic peptides similar in composition to LMWCr and apoLMWCr. The sequence
pEEEEGDD can bind 4 chromic ions per peptide as apoLMWCr does, while the other sequences
examined only bind two chromic ions.
Studies to further elucidate the structure of LMWCr are ongoing.
ii
DEDICATION
To my mother and father, who give my life and grow my soul
To my brother, who walks with me and cheers me
To the Chens and Yangs, who encourage me and support me
To Juan, who I love and loves me
And to
All my friends
iii
LIST OF ABBREVIATIONS AND SYMBOLS
α
Alpha
AA
Amino acid
Apo
Metal-free
acac
Acetylacetonate
β
Beta
B
Racah parameter
BSA
Bovine serum albumin
C
Carbon
C (amino acid)
Cysteine
Ci
Curie
CID
Collision-induced dissociation
Cl
Chloride
cm
Centimeter
cm-1
Reciprocal centrimeter
Co
Cobalt
Cr
Chromium
[Cr]
Concentration of chromium
Cr(III)
Chromic ions
Cr2O72+
Chromate
iv
CrCl3
Chromium chloride
D (amino acid)
Aspartic acid
Da
Dalton
DEAE
Diethylaminoethyl cellulose
dH2O
Deionized water
Dq
Crystal field splitting
D2O
Deuterium oxide
ε
Extinction coefficient
E (amino acid)
Glutamic acid
EDTA
Ethylenediamine tetraacetate
EPR
Electron paramagnetic resonance
ESI
Electrospray ionization
g
Gram
G (amino acid)
Glycine
GTF
Glucose tolerance factor
H
Hydrogen
HCl
Hydrochloric acid
HDL
High density lipoprotein cholesterol
Hepes
N-2-hydroxyethylpiperizine-N’-2-ethanesulfonic acid
H2O
Water
H2O2
Hydrogen peroxide
HPLC
High-performance liquid chromatography
IGF-1
Insulin-like growth factor-1
v
IR
Insulin receptor
IRS-1
Insulin receptor substrate-1
K
Kelvin
K2Cr2O7
Potassium dichromate
kg
Kilogram
kDa
Kilo-Daltons
L
Liter
LC-MS
Liquid chromatograph-mass spectrometers
LDL
Low density lipoprotein cholesterol
LMWCr
Low-molecular-weight chromium-binding substance
M
Molar
M-1
Reciprocal molar
MALDI
Matrix-assisted laser desorption/ionization
mg
Milligram
MHz
Megahertz
min
Minute
mL
Milliliter
mM
Millimolar
MS
Mass Spectrometry
MS/MS
Tandem mass spectrometry
MW
Molecular weight
n
Hill coefficient
NaCl
Sodium chloride
vi
NaOH
Sodium Hydroxide
NH4+
Ammonium
NH4OAc
Ammonium acetate
nm
Nanometer
NMR
Nuclear magnetic resonance
O
Oxygen
P (amino acid)
Pyro-
PMSF
phenylmethylsulfonyl fluoride
p-NPP
Para-nitrophenyl phosphate
pic
Picolinate
ppm
Parts per million
PSD
Post-source decay
rpm
Revolutions per minute
SDS
Sodium dodecyl sulfate
TFA
Trifluoroacetic acid
Tris
Tris-(hydroxymethyl) aminomethane
µg
Microgram
µL
Microliter
µM
Micromolar
UV
Ultraviolet
UV-vis
Ultraviolet-visible
×
Times
y
Fraction of macromolecule saturated
vii
Zn
Zinc
°C
Degree Celsium
%
Percent
viii
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. John B. Vincent, for his encouragement, patience,
and guidance throughout my entire graduate career. Without his advice and help, I could not
have completed my Ph.D. studies.
I am grateful to Dr. Stephen A. Woski for teaching me many research techniques and
allowing me to use his laboratory. When I was facing a funding problem, he and Dr. Vincent
tried their best to help me over the hard time.
I am indebted to Dr. Carolyn J. Cassady for helping me on mass spectrometry. All
progress on this project could not have been made without her solid support.
I am appreciative of Dr. Janis M. O'Donnell and Dr. Laura S. Busenlehner for serving on
my committee and assisting me on biological problems.
I would like to express my sincere thanks to certain members of my research group and
Dr. Cassady’s group, past and present. I would like to recognize Dr. Michael J. Hatfield, who
first taught me the basic laboratory skills necessary for this project. For assistance and friendship,
I would like to acknowledge Dr. Dontarie Stallings, Nicholas Rhodes, DeAna McAdory, Afirahs
Evol and Dr. Sarmistha Halder. In this joint project, I would like to acknowledge Dr. Junjie Gao
and Heather M. Watson, who spent a lot of time on sequencing LMWCr. I would like to express
my thanks to Dr. Qiaoli Liang and Dr. Ken Belmore for their assistance on LC-MS and NMR,
respectively. I also want to thank Yinghua Chen, who contributed some ideas to the future work
section.
ix
This research would not have been achieved without the supports of my friends and
fellow graduate students and of course my family, who never stopped encouraging me with their
love and understanding.
Thank you!
x
CONTENTS
ABSTRACT .................................................................................................................................... ii
DEDICATION ............................................................................................................................... iii
LIST OF ABBREVIATIONS AND SYMBOLS .......................................................................... iv
ACKNOWLEDGMENTS ............................................................................................................. ix
LIST OF TABLES ....................................................................................................................... xiv
LIST OF FIGURES .......................................................................................................................xv
LIST OF ILLUSTRATIONS ....................................................................................................... xvi
OVERALL INTRODUCTION........................................................................................................1
Chapter 1
Introduction: LMWCr ................................................................................................2
1.1 Chromium and Diabetes..................................................................................................... 2
1.2 Low-Molecular-Weight Chromium-Binding Substance (LMWCr)................................... 4
1.3 Synthetic Chromium Compounds....................................................................................... 6
1.4 Summary ......................................................................................................................... 11
REFERENCES........................................................................................................................... 12
Chapter 2
Isolation and Characterization of Low-Molecular-Weight-Chromium
Oligopeptide from Non-mammal and Human Urine ...............................…............15
2.1 Introduction........................................................................................................... 15
2.2 Instrumentation..................................................................................................... 16
2.3 Materials ............................................................................................................... 16
xi
2.4 Methods................................................................................................................. 17
2.4.1 Chromium Concentration Determination.................................................. 17
2.4.2 Protein and Oligopeptide Concentration Determination.......................................... 17
2.4.3 Isolation of LMWCr from Chicken and Alligator Liver ………………................ 18
2.4.4 Isolation of LMWCr from Human Urine................................................................. 20
2.4.5 Molecular Weight Determination............................................................................ 20
2.4.6 Phosphatase Activity Assays .................................................................................. 21
2.5 Results and Discussion..................................................................................................... 21
2.5.1 Isolation and Purification......................................................................................... 21
2.5.2 Physical Characterization........................................................................................ 36
2.5.3 Electronic Spectroscopic studies ……………........................................................ 36
2.5.4 Electron Paramagnetic Resonance........................................................................... 38
2.5.5 Biological activity ................................................................................................... 46
2.6 Conclusions....................................................................................................................... 46
REFERENCES................................................................................................................. 48
Chapter 3
The Binding of Trivalent Chromium to Candidate Sequences of LMWCr ............51
3.1 Introduction...................................................................................................................... 51
3.2 Instrumentation....................................................................................................... 53
3.3 Materials ......................................................................................................................... 53
3.3.1 Synthetic Peptides................................................................................................... 53
3.3.2 Other Chemicals ...................................................................................................... 53
3.4 Methods............................................................................................................................. 54
3.4.1 Physical Characterization of Cr-peptide Complex Studies...................................... 54
xii
3.4.2 ApoLMWCr and Synthetic Peptides Studies........................................................... 54
3.4.3 Miscellaneous......................................................................................................... 55
3.5 Result and Discussion ...................................................................................................... 55
3.5.1 Quantitative Analysis of Cr Bound to Peptides ...................................................... 55
3.5.2 Effect of Cr(III) concentration ............................................................................... 58
3.5.3 UV/Vis spectra of the Cr(III) loaded Synthetic Peptide ......................................... 70
3.6 Conclusions...................................................................................................................... 84
REFERENCES................................................................................................................. 85
Chapter 4
Sequence LMWCr by MS ......................................................................................87
4.1 Introduction...................................................................................................................... 87
4.2 Instrumentation................................................................................................................. 90
4.3 Materials........................................................................................................................... 91
4.4 Methods..................................................................................................................... 91
4.4.1 Graphite Powder Micro column............................................................................... 91
4.4.2 Immunization.......................................................................................................... 92
4.5 Results and Discussion..................................................................................................... 92
4.5.1 MALDI-TOF MS Studies ..................................................................................... 92
4.5.2 Analysis of LMWCr using LCMS with ESI............................................................ 93
4.5.3 Immunization and bioinformatics ........................................................................... 95
4.6 Conlusion and Future Work........................................................................................... 106
REFERENCES ......................................................................................................................... 108
xiii
LIST OF TABLES
2.1 Purification of LMWCr from chicken, alligator liver and human urine .............................. 34
2.2 Comparison of the amino acid cmposition of LMWCr’s and the Cr:peptide ratios from
different sources ................................................................................................................... 37
2.3 Comparison of visible spectra of bovine, chicken, alligator and human urine LMWCr ….. 42
3.1 Temperature program of atomic adsorption assay for Cr(III) in solution ........................... 56
3.2 Hill plot constants of apoLMWCr and synthetic peptides. ................................................. 76
3.3
Langmuir isotherm parameters of apoLMWCr and synthetic peptides.............................. 78
4.1 ELISA results of rabbit’s serum immunization with synthetic peptides as
immunogens…………………………….………………………………………………....105
xiv
LIST OF SCHEMES
1.1
Proposed mechanism for the activation of insulin receptor activity by
low-molecular-weight chromium-binding substance (LMWCr) in response to
insulin (Adapted from Ref. 22). ............................................................................................. 8
1.2
Proposed mechanism for the movement of Cr from blood to apoLMWCr
responding to increases in insulin concentration (Adapted from Ref. 27). ........................... 9
3.1 Flow chart for study of Cr(III) binding by peptides. ........................................................... 57
4.1 Peptide fragmentation notation using the scheme of Roepstorff and Fohlman. .................. 88
xv
LIST OF FIGURES
1.1. Structure of Cr3, the cation [Cr3O(O2CCH2CH3)6(H2O)3]+ ........................................... 10
2.1. Elution profile of alligator liver Cr-containing fractions on first DEAE column. ............... 23
2.2. Elution profile of alligator liver Cr-containing fractions on second DEAE column. .......... 24
2.3. Elution profile of alligator liver Cr-containing fractions on Sephadex G-50 column. ........ 25
2.4. Elution profile of alligator liver Cr-containing fractions on Sephadex G-25 column. ........ 26
2.5. Elution profile of alligator liver Cr-containing fractions on Sephadex G-15 column. ........ 27
2.6. Elution profile of alligator liver Cr-containing fractions on HPLC using a Shodex OH
PAK column......................................................................................................................... 28
2.7. Elution profile of human urine Cr-containing fractions on first DEAE column. ................ 29
2.8. Elution profile of human urine Cr-containing fractions on second DEAE column. ............ 30
2.9. Elution profile of human urine Cr-containing fractions on Sephadex G-25 column. .......... 31
2.10. Elution profile of human urine Cr-containing fractions on Sephadex G-15 column. .......... 32
2.11. Elution profile of human urine Cr-containing fractions on HPLC using a Shodex OH
PAK column......................................................................................................................... 33
2.12. SDS-PAGE gel electrophoresis of chicken, alligator and human urine LMWCr................ 35
2.13. (a) Ultraviolet spectrum of chicken liver LMWCr in 50 mM ammonium acetate,
pH 6.5. [Cr] = 0.2 mM; (b) visible spectrum of chicken liver LMWCr in 50 mM
ammonium acetate, pH 6.5. [Cr] = 1.1 mM. ........................................................................ 39
2.14. [a] Ultraviolet spectrum of alligator liver LMWCr in 50 mM ammonium acetate,
pH 6.5. [Cr] = 0.6 mM;Visible spectrum of alligator liver LMWCr in 50 mM
ammonium acetate, pH 6.5. [Cr] = 4.7 mM. ........................................................................ 40
xvi
2.15. [a] Ultraviolet spectrum
s
of human urinee LMWCr inn 50 mM am
mmonium aceetate,
pH 6.5. [Cr] = 0.5
0 mM; [b] Visible specctrum of alliigator liver LMWCr
L
in 50
5 mM
amm
monium aceetate, pH 6.5, [Cr] = 2.8 mM.
m ............................................................................ 41
2.16. X-bband EPR sp
pectrum of chhicken liver and alligatoor LMWCr inn 50 mM am
mmonium
aceetate buffer, pH
p 6.5. (Topp) Chicken liver LMWC
Cr. [Cr] = 15
1 mM. Insttrumental
parrameters: mo
odulation frequency, 1000 kHz; moduulation ampliitude, 10.59 G; time
connstant, 20.48 ms; sweep time,
t
83.89 s; field centeer, 4000 G; sweep
s
widthh, 7600 G;
freqquency, 9.63
327 GHz; annd power, 2.00 mW. (Botttom) Alligatoor liver LMW
WCr.
[Crr] = ~15 mM
M. Instrumenttal parameteers: modulatiion frequenccy, 100 kHz;
modulation amp
plitude, 10.559 G; time coonstant, 20.448 ms; sweepp time, 83.89 s; field
cennter, 4000 G;; sweep widtth, 7600 G; frequency,
f
9
9.6308
GHz; and power, 2.0 mW ......... 44
2.17. X-bband EPR sp
pectrum of urine
u
LMWC
Cr in 50 mM ammonium acetate bufffer,
pH 6.5. [Cr] = ~13.5
~
mM. T = 77 K, Innstrumental parameters:
p
M
Modulation
G frequencyy, 9.047 GHzz and power, 2.0 mW. time constantt = 0.5 s;
ampplitude 5.0 G;
gainn = 5 x 104; 2 scans.............................................................................................................. 45
3.1
Eluution profile of Cr-oligoppeptide appliied to Sephaadex G-10 coolumn (260 nm).
n ................ 59
3.2. UV spectra of sp
pecies formeed upon the addition of varying
v
quanntities of chrromic ions
to the peptide EDGEECDC
CGE. Numbbers in inset correspond to
t Cr:peptidee ratios.
[peeptide] = 0.1
18 mM. ............................................................................................................... 60
3.3. UV spectra of sp
pecies formeed upon the addition of varying
v
quanntities chrom
mic ions to
peeptide DGEE
ECDCGED. Numbers in inset correspond to Cr:ppeptide ratioos. [peptide] =
0.118 mM. ................................................................................................................................. 61
3.4. Sorpption isotherrm of Cr(III)) ion on apoL
LMWCr andd synthetic peptides.
p
Vollume of
addsorption medium: 200 mL,
m temperatture: 4 °C, adsorption
a
tim
me: 12 h, iniitial pH: 7.4 ... 62
3.5. Lanngmuir linearr isotherm off Cr(III) adsorbed on apooLMWCr, Volume
V
of addsorption
meedium: 200 ml
m , temperaature: 4 °C, adsorption
a
tiime: 12 h, innitial pH: 7.44. ...................... 65
3.6. Lanngmuir linearr isotherm off Cr(III) adsorbed on ED
DGEECDCG
GE, Volume of adsorptioon
meedium: 200 ml
m , temperaature: 4 °C, adsorption
a
tiime: 12 h, innitial pH: 7.44. ...................... 66
3.7. Lanngmuir linearr isotherm off Cr(III) adsorbed on DG
GEECDCGE
EE, Volume of adsorptioon
meedium: 200 ml
m , temperaature: 4 °C, adsorption
a
tiime: 12 h, innitial pH: 7.44. ...................... 67
3.8. Lanngmuir linearr isotherm off Cr(III) adsorbed on pE
EEEEGDD, Volume
V
of adsorption
a
meedium: 200 ml
m , temperaature: 4 °C, adsorption
a
tiime: 12 h, innitial pH: 7.44. ...................... 68
3.9. Hill plot of chrom
mic ion bindding to apoL
LMWCr. ....................................................................... 72
omic ion binnding to pepttide EDGEE
ECDCGE. ................................................ 73
3.10. Hill plot of chro
3.11. Hill plot of chro
omic ion binnding to pepttide DGEEC
CDCGEE. ................................................ 74
xvii
3.12. Hill plot of chromic ion binding to synthetic peptide pEEEEGDD..................................... 75
3.13. Langmuir isotherm for Cr(III) uptake by apoLMWCr and synthetic peptides at 4 °C........ 77
3.14. Langmuir isotherm (BM0.5 model) of Cr(III) adsorbed on apoLMWCr, Volume of
adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4. .... 79
3.15. Langmuir isotherm (BM0.5 model) of Cr(III) adsorbed on EDGEECDCGE, Volume
of adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH:7.4. . 80
3.16. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-EDGEECDCGE complex.
[Cr] = 0.03 mM. ................................................................................................................... 81
3.17. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-DGEECDCGEE complex.
[Cr] = 0.04 mM. ................................................................................................................... 82
3.18. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-oligopeptide pEEEEGDD. .......... 83
4.1. Signals with m/z ~804 peak appear in the spectra of all treated LMWCr’s with GP
column in negative mode MALDI-TOF MS. ...................................................................... 97
4.2. MALDI-TOF PSD of m/z 804 ion of Bovine LMWCr in 70% ACN/0.1%
TFA/10% 4HCCA ............................................................................................................... 98
4.3. MALDI-TOF PSD of m/z 804 ions of alligator, bovine, chicken, human urine
and synthetic peptides, pEEEEGDD and pEEEGEDD. ...................................................... 99
4.4. Elution profile on Zorbax 5B-C18 column in dH2O and acetonitrile (linear gradient).
(top) Total Ion Concentration (TIC), (middle) UV chromatogram at 260 nm, and
(bottom) spectra of MS in sharp peak area. ....................................................................... 100
4.5. ESI-MS (top) of bovine LMWCr treated with GP processing, (middle) of pEEEEGDD
and (bottom) of pEEEGEDD. ............................................................................................ 100
4.6. Low CID spectra of (top) MS2 of m/z ~401 and (bottom) MS3 of m/z ~391 from
bovine LMWCr samples. Double Charged peaks are marked with "2-". ......................... 100
4.7. Low CID ESI-MS/MS (top) of bovine LMWCr treated with GP processing,
(middle) of pEEEEGDD and (bottom) of pEEEGEDD ................................................... 100
4.8. Low CID ESI-MS/MS/MS (top) of bovine LMWCr treated with GP processing,
(middle) of pEEEEGDD and (bottom) of pEEEGEDD ................................................... 100
xviii
OVERALL INTRODUCTION
The interaction between Cr ions and bio-molecules plays an important role in
mammalian glucose and lipid metabolism. Understanding the molecular-level mechanism of how
Cr ions in vivo affect insulin and LDL cholesterol levels will help confirm if Cr supplements can
improve symptoms of type 2 diabetes and related conditions. This dissertation is composed of
four chapters. In chapter 1, the relationship of Cr and diabetes II is discussed. Mechanisms of
how Cr, in the biological form of low-molecular-weight Cr (LMWCr), works on insulin sensitive
cells and how Cr is transferred in vivo are presented. Some Cr supplements on the market are
also discussed. In chapter 2, based on the hypothesis presented in chapter 1, modified methods
of isolation of LMWCr from animal other than mammals and from human urine are presented. In
chapter 3, based on the sequences of some for LMWCr, Cr-binding constants of some synthetic
peptides are examined and compared to those of apoLMWCr. Two mathematical models,
Langmuir isotherm and Hill plot, were applied to process data. In chapter 4, a novel method is
introduced to generate a MS signal of LMWCr using a home-made graphite micro-column.
Sequences deduced from results of MALDI-TOF PSD and ESI MS CID was discussed. Finally,
based on results of chapters 3 and 4, the prospect of finalizing the LMWCr sequence and
structure is discussed.
1
1.
CHAPTER 1
INTRODUCTION: LMWCr
1.1
Chromium and Diabetes
Recognition of chromium (Cr) as a potentially essential trace element for mammals arose
from apparently Cr-deficient rats studied by Walter Mertz and coworkers in the 1950s. The rats
that were fed a Torula yeast diet, which was proposed to be chromium deficient, developed a
decreased ability to remove blood glucose after being given an intravenous glucose load. Cr-rich
materials isolated from acid-hydrolyzed porcine kidney powder and from Brewer’s yeast were
found to reverse the glucose intolerance; the active agent in these was named glucose tolerance
factor (GTF).1 Subsequently, Cr(III) in the GTF was proposed to be necessary agent for the
maintenance of normal glucose tolerance.2 These results have not stood the test of time. The
rats were kept in metal cages and could have obtained Cr from chewing on the metal; the Cr
content of the diet was not actually determined. Given that stainless steel is ~40% Cr, the rats
were probably not Cr deficient. Hence, whether Cr is an essential element is currently a matter
of debate.
Type 2 diabetes (non-insulin-dependent diabetes mellitus, NIDDM) is one of the major
categories of the disorders of glycemia or glucose tolerance; the symptoms of type 2 diabetes
have been proposed to reflect a relationship between Cr and insulin action. Insulin action impacts
on glucose homeostasis through insulin receptor (IR), a heterotetrameric glycoprotein consisting
2
of two extracellular α-subunits (135 kDa) and two transmembrane β-subunits (95 kDa) with
intrinsic protein kinase activity. Insulin binding to the α-subunits and activates the tyrosine
kinase of the β-subunits via the induction of a conformational change of the insulin receptor.
This change juxtaposes the intracellular kinase domains at the correct distances and orientations
for transphosphorylation. In this conformation, IR can phosphorylate tyrosine residues on some
immediate substrates, including insulin receptor substrate (IRS) proteins that provide specific
docking site for other signaling proteins containing SH2 domains. These events lead to the
activation of downstream signaling molecules,
including PI 3-Kinase (PI 3-K), that
consequentially regulate trafficking of the glucose transporter GLUT4 between the plasma
membrane and one or more intracellular compartments.3
Type 2 diabetes results from an impairment of insulin action in hepatic and peripheral
tissues, especially muscle tissue and adipocytes, and eventually from a defect in insulin secretion.
A post receptor (or at least after insulin binding to the receptor) defect is present, causing
resistance to the stimulatory effect of insulin on glucose use. As a result, the beta cells of the
pancreas produce more insulin to compensate for the resistance. Production of insulin generates
oxidative damage to the cells, which when constantly producing insulin are unable to repair the
damage leading to cell death. The body then cannot produce enough insulin to compensate and a
relative insulin deficiency develops in addition to the insulin insensitivity. Treatment of animals
and humans with Cr supplements has been reported in lead to improvements in diabetes
symptoms and led many investigators to attempt to elucidate the mechanism whereby Cr
improves insulin insensitivity, possibly by participating in the insulin signaling pathway.4,5 In
support of this theory, Morris and coworkers6 have shown that plasma Cr levels decrease as the
result of increases in blood insulin concentration following an oral glucose load; further
3
chromium loss continued with a subsequent infusion of insulin. Within 90 minutes blood after
the increase of blood insulin, Cr concentration started to recover.6 Thus, Cr movement appears to
be linked to insulin action.
The biochemistry of chromium’s potential role in IR activation is poorly understood. But,
hypotheses include direct interaction of Cr with insulin receptor or a role of Cr in increasing IR
number, as well as changing cell membrane properties to impact the function of membrane
proteins mediating insulin action.7,8,9
1.2
Low-Molecular-Weight Chromium-Binding Substance (LMWCr)
A possible agent of Cr action is LMWCr, low-molecular-weight chromium-binding
substance.
LMWCr is a Cr-containing oligopeptide of about 1500 Da molecular weight.
LMWCr was first isolated by the Japanese toxicologists Yamamoto and Wada from bovine
colostrum.10 (Colostrum is the fresh milk produced soon after birth). Afterward, its analogs
were isolated and purified or partially purified from the livers of rabbits, dogs, and mice.11,12,13
LMWCr from the various sources is comprised solely or almost solely of only four amino acids:
glycine, cysteine, glutamate and aspartate. The molar ratio of chromic ion to oligopeptide in
LMWCr from rabbit and bovine liver is 4 to 1.
LMWCr has been found to increase the metabolization of glucose to produce CO2 and
total lipids in the presence of insulin by rat adipocytes.10 The similar degree of stimulation at
half-maximal insulin concentration suggests LMWCr may play a role after insulin binds
externally to the insulin receptor.14 The stimulation was dependent on the chromium content of
LMWCr.15
Large quantities of LMWCr become available for spectroscopic, magnetic and kinetic
studies after the Vincent group developed a procedure to purify LMWCr from bovine liver.16
4
Bovine LMWCr tightly (Ka ~ 1021 M-4) and highly cooperatively (Hill coefficient, n=3.47) binds
4 chromic ions such that only apoLMWCr (no Cr bound) and holoLMWCr (4 Cr bound) co-exist
in solution. ApoLMWCr can remove chromium from Cr2-transferrin at near neutral pH.17
What role (if any) does LMWCr play in vivo that requires Cr? A detoxification function
for LMWCr proposed by Yamamoto and Wada is unlikely to be its primary one, since injection
of Cr into mice did not stimulate the production of LMWCr.13 Bovine LMWCr has been
demonstrated in vitro to activate a membrane phosphotyrosine phosphatase and to amplify the
IR’s tyrosine kinase activity as much as 8-fold in the presence of 100 nM insulin using adipocyte
membrane fragments, while little activation of kinase activity is observed with using apoLMWCr
or without insulin present.18,19 LMWCr is maintained in the cytosol of insulin-dependent cells
almost in its apo-(metal free) form.6 As Cr in blood is taken up by insulin-dependent cells in
response to a increase of insulin concentration (which results from increase in carbohydrate
levels), a role for LMWCr in binding this Cr and then participating in enhancing insulin
signaling would seem possible.6,20,21 Based on the above studies, a mechanism of action for
LMWCr was proposed by the Vincent group where LMWCr is part of a unique
autoamplification system for insulin signaling (Scheme 1.1). In this proposed mechanism, in
response to increases in blood insulin concentrations, insulin binds to IR, initiating a
conformational change which affects intracellular receptor tyrosine kinase activation. Increases
of the insulin level in the blood also results in moving Cr from the blood to insulin-dependent
cells, where apoLMWCr is loaded with Cr to produce holoLMWCr. The structure of
holoLMWCr allows it to bind to the β-subunits of IR to keep the IR’s active conformation from
reversing, amplifying the kinase activity. Bound holoLMWCr dissociates from IR when the
signal is to be turned off as insulin levels drop (as proteases degrade the insulin). Thus, LMWCr
5
was named as chromodulin due to its potentially similar function to that of the calcium signal
protein calmodulin. Calmodulin binds to kinases and phosphatases activiating after the protein
binds four equivalents of calcium.
Cr transport into human or rat insulin-dependent tissues is significantly enhanced by
glucose, suggesting that Cr may translocate from the blood compartment to insulin-sensitive
cells.6 More than 80% of plasma Cr is bound to transferrin (Tf) after an intravenous Cr(III) load.
The different binding constants between apoLMWCr and Tf make it possible for Cr transfer
from Tf to apoLMWCr. In response to insulin, Cr is transported in the form of Cr-Tf to insulindependent cells, such as hepatocytes and skeletal muscle cells, and subsequently lost in the urine
as holoLMWCr.22 Insulin stimulated movement of Cr from Cr-Tf in the blood plasma to tissues
then to urine has been observed to occur in less than 30 minutes.23 A Cr transport mechanism
(Scheme 1.2) has been proposed based on relationship between insulin action and LMWCr
movement.
1.3
Synthetic Chromium Compounds
Cr exists in several valence states. Among them, only trivalent and hexavalent are
appreciably stable in air and water. However, hexavalent has been found to be genotoxic and
carcinogenic. The trivalent state of Cr (Cr(III)) becomes the only potentally biologically active
form, possibly as LMWCr. Commercial Cr supplements are available with trivalent Cr as Cr
chloride (CrCl3), Cr picolinate (Cr(Pic)3), or Cr nicotinate, whose formula has not been firmly
established. Other forms of Cr(III) are often proposed for use as Cr supplements but have not
made the commercial market.
The potentially bioactive form of Cr(III) LMWCr contains a multinuclear Cr(III)
assembly. The results of a combination of X-ray absorption and electron paramagnetic resonance
6
(EPR) spectroscopies and variable-temperature magnetic susceptibility measurements suggest
that bovine holoLMWCr possesses an antiferromagnetically coupled trinuclear assembly that
weakly interacts with a fourth Cr(III) center.24 The trinuclear assembly is bridged by oxygenbased ligation and supported by carboxylates provided by the polypeptide. Based on the evidence
of
multinuclear
core
in
LMWCr,
the
trinuclear
chromium
carboxylate
complex
[Cr3O(O2CCH2CH3)6(H2O)3]+ (Cr3) (Figure 1.1) was found to be a functional biomimetic and
proposed for use as nutritional supplement and therapeutic agent.
Cr3 can activate the kinase of a catalytically active fragment of the β subunit of human IR
up to three-fold in vitro.25 Studies on healthy and Zucker obese rats (an early stage type 2
diabetes model) over 24 weeks of intravenous administration of Cr3 (20 µg/kg body mass)
revealed Cr3 lowered fasting plasma total cholesterol, triglycerides, insulin, and low-density
lipoprotein (LDL) cholesterol concentrations, suggesting Cr3 increases insulin sensitivity, in
contrast to CrCl3 and Cr picolinate that had no effect on any of these variables in healthy rats.26
Similar results were obtained in a study in which the doses of the biomimetic compound were
given orally.27 Cr3 has more than a 10-fold greater degree of absorption over those of Cr
picolinate and CrCl3 at either nutritionally relevant or a pharmacologically relevant doses (at
least 40-60% absorption versus 0.5-2% absorption).28
Cr(pic)3 is a very popular nutritional supplement and is being tested as a therapeutic for
the treatment of adult-onset diabetes. However, some studies show Cr(pic)3 can produce reactive
oxygen species, which can cleave DNA in vitro.29 A large percentage of female fruit flies feed a
diet containing Cr(pic)3 at levels equivalent to a human taking a Cr supplement (200 g Cr per day)
developed sterility, and their offspring possessed increased levels of lethal mutations and delayed
development, suggesting potential DNA damage from the supplement.24 Skeletal effects were
7
Scheme 1.1. Proposed mechanism for the activation of insulin receptor activity by lowmolecular-weight chromium-binding substance (LMWCr) in response to insulin (Adapted
from Ref. 22).
8
Scheme 1.2. Proposed mechanism for the movement of Cr from blood to apoLMWCr
responding to increases in insulin concentration (Adapted from Ref. 27).
9
R=C2H5, L=H2O
Figure 1.1. Structure of Cr3, the cation [Cr3O(O2CCH2CH3)6(H2O)3]+
10
observed in offspring of pregnant mice exposed to Cr(pic)3 in their food30. These negative effects
are not found studies with CrCl3 or Cr3.31
1.4
Summary
Scientists have been debating for more than 50 years whether chromium is an essential
element for mammals. Several models based on inconsistent data and methods attempted to
explain chromium’s relationship to glucose homeostasis and its effects on diabetic symptoms at a
molecular level. The hypothesis represented by LMWCr holds promise, but the potential
mechanism of action lacks supporting in vivo evidence.
11
REFERENCES
(1)
Schwarz, K.; Mertz, W. Archives of Biochemistry and Biophysics 1957, 72, 515-518.
(2)
Schwartz, K.; Mertz, W. Archives of Biochemistry and Biophysics 1959, 85, 292-295.
(3)
Saltiel, A. R.; Kahn, C. R. Nature 2001, 414, 799-806.
(4)
Anderson, R. A. Diabetes & Metabolism 2000, 26, 22-27.
(5)
Cefalu, W. T.; Wang, Z. Q.; Zhang, X. H.; Baldor, L. C.; Russell, J. C. Journal of
Nutrition 2002, 132, 1107-1114.
(6)
Morris, B. W.; Gray, T. A.; Macneil, S. Clinical Science 1993, 84, 477-482.
(7)
Mertz, W. Physiol. Rev. 1969, 49, 163-239.
(8)
Anderson, R. A.; Polansky, M. M.; Bryden, N. A.; Bhathena, S. J.; Canary, J. J.
Metabolism-Clinical and Experimental 1987, 36, 351-355.
(9)
Chen, G.; Liu, P.; Pattar, G. R.; Tackett, L.; Bhonagiri, P.; Strawbridge, A. B.; Elmendorf,
J. S. Molecular Endocrinology 2006, 20, 857-870.
(10)
Yamamoto, A.; Wada, O.; Suzuki, H. Journal of Nutrition 1988, 118, 39-45.
(11)
Cohen, G. B.; Ren, R. B.; Baltimore, D. Cell 1995, 80, 237-248.
12
(12)
Anderson, R. A. In Trace Elements in Human and Animal Nutrition 5th ed.; Mertz, W.,
Ed.; Academic Press, Inc.: New York, 1987, p 225-244.
(13)
Yamamoto, A.; Wada, O.; Ono, T. Journal of Inorganic Biochemistry 1984, 22, 91-102.
(14)
Vincent, J. B. Journal of Nutrition 1994, 124, 117-118.
(15)
Yamamoto, A.; Wada, O.; Manabe, S. Biochemical and Biophysical Research
Communications 1989, 163, 189-193.
(16)
Davis, C. M.; Vincent, J. B. Abstracts of Papers of the American Chemical Society 1996,
211, 585-INOR.
(17)
Sun, Y.; Ramirez, J.; Woski, S. A.; Vincent, J. B. Journal of Biological Inorganic
Chemistry 2000, 5, 129-136.
(18)
Davis, C. M.; Sumrall, K. H.; Vincent, J. B. Biochemistry 1996, 35, 12963-12969.
(19)
Davis, C. M.; Vincent, J. B. Biochemistry 1997, 36, 4382-4385.
(20)
Morris, B. W.; Blumsohn, A.; Macneil, S.; Gray, T. A. American Journal of Clinical
Nutrition 1992, 55, 989-991.
(21) Morris, B. W.; MacNeil, S.; Stanley, K.; Gray, T. A.; Fraser, R. J Endocrinol 1993, 139,
339-345.
(22)
Clodfelder, B. J.; Emamaullee, J.; Hepburn, D. D. D.; Chakov, N. E.; Nettles, H. S.;
Vincent, J. B. Journal of Biological Inorganic Chemistry 2001, 6, 608-617.
(23)
Clodfelder, B. J.; Upchurch, R. G.; Vincent, J. B. Journal of Inorganic Biochemistry
2004, 98, 522-533.
(24)
Jacquamet, L.; Sun, Y.; Hatfield, J.; Gu, W.; Cramer, S. P.; Crowder, M. W.; Lorigan, G.
A.; Vincent, J. B.; Latour, J.-M. Journal of the American Chemical Society 2003, 125,
774-780.
(25)
Davis, C. M.; Royer, A. C.; Vincent, J. B. Inorganic Chemistry 1997, 36, 5316-5320.
13
(26)
Sun, Y. J.; Mallya, K.; Ramirez, J.; Vincent, J. B. Journal of Biological Inorganic
Chemistry 1999, 4, 838-845.
(27)
Clodfelder, B. J.; Gullick, B. M.; Lukaski, H. C.; Neggers, Y.; Vincent, J. B. Journal of
Biological Inorganic Chemistry 2005, 10, 316-316.
(28)
Olin, K. L.; Stearns, D. M.; Armstrong, W. H.; Keen, C. L. Trace Elements and
Electrolytes 1994, 11, 182-186.
(29)
Speetjens, J. K.; Collins, R. A.; Vincent, J. B.; Woski, S. A. Chemical Research in
Toxicology 1999, 12, 483-487.
(30)
Bailey, M. M.; Boohaker, J. G.; Sawyer, R. D.; Behling, J. E.; Rasco, J. F.; Jernigan, J. J.;
Hood, R. D.; Vincent, J. B. Birth Defects Research Part B-Developmental and
Reproductive Toxicology 2006, 77, 244-249.
(31)
Stallings, D. M.; Hepburn, D. D. D.; Hannah, M.; Vincent, J. B.; O'Donnell, J. Mutation
Research-Genetic Toxicology and Environmental Mutagenesis 2006, 610, 101-113.
14
2.
CHAPTER 2
ISOLATION AND CHARACTERIZATION OF LOW-MOLECULAR-WEIGHT
CHROMIUM OLIGOPEPTIDE FROM NON-MAMMALIAN SOUCRES
AND HUMAN URINE
2.1
Introduction
Although its function is not known with certainty, low-molecular-weight chromium-
binding substance (LMWCr) has been proposed to operate as a factor in chromium transport and
potentiating insulin signals in mammals. To date studies involving LMWCr have been limited to
mammals, and LMWCr has only have been isolated from several mammalian sources.1 If
LMWCr is involved with the in vivo activation of the kinase activity of insulin receptor and thus
glucose metabolism, LMWCr might be expected to be present in any animal that utilizes insulin.
Notably, insulin of turkey and alligator lead to similar glycemic responses as bovine insulin in
fasted alligators, suggesting a similarity in the activation of insulin receptor among all three
classes of animals: reptiles, aves, and mammals.2 Compared to mammals, birds are insulinresistant. However, the basic machinery for insulin activation is present in the liver.3 Since
tissues from chicken and American alligator are available in bulk, they were chosen to represent
these two classes of animals.
The transportation of Cr in the body has been followed from its binding to transferrin in
the bloodstream to its release from the body in the urine.4 LMWCr appears to be involved in
transporting Cr from the tissues to the urine.
Based on the composition of LMWCr, a
tetrapeptide comprised of glutamate, aspartate and glycine notably is found in urine from
15
lipoatrophic diabetes patients.5 One outstanding question in this process is whether the LMWCr
(firmly established for at least mammalian liver and kidneys) is the same low molar mass species
which contains Cr in the urine; if true, this would unequivocally establish a role for LMWCr in
the transport and removal of Cr from the body. This was first assumed in the work by Wada and
coworkers,6 and several subsequent groups have made similar assumptions. One problem in
conducting studies on the form of Cr in urine is the low concentration of Cr in urine, ~0.5 ppb,
although large quantities of peptides (1–4 g) are excreted in human urine each day.6
This chapter describes methods developed for the isolation of LMWCr from chicken and
alligator liver, as well as from human urine in milligram quantities for spectroscopic studies.
2.2
Instrumentation
A Sorvall RC-5B Superspeed centrifuge from Dupont Instruments with a GS-3 rotor was
used for isolation of LMWCr. Electronic spectra were collected on a Hewlett-Packard 8451A or
a Beckman Coulter DU 800 spectrophotometer. Fluorescence measurements were obtained on a
Jobin Yvon FluoroMax-3 fluorescence spectrophotometer. For high performance liquid
chromatography, HPLC, a Shodex OH PAK B-803 series size exclusion column (8 × 100 mm)
and 50 µL injections were utilized. Electron paramagnetic resonance (EPR) spectra of chicken
and alligator LMWCr were obtained courtesy of the Medical College of Wisconsion’s National
Biomedical EPR Center, while human urine LMWCr was examined on a Bruker ELEXSYS E580 pulsed-EPR spectrometer. Amino acid analyses were performed by the Protein and
Separation Analysis Laboratory at Purdue University.
2.3
Materials
Phenylmethlysulfonyl fluoride (PMSF) and ammomium acetate (NH4OAc) were obtained
from Research Organics, Inc. Benzamide, pepstatin A, insulin A, bradykinin and glycine were
16
obtained from Sigma. Diphenylcarbazide, hydrogen peroxide (H2O2), and other common
reagents were purchased from Fisher. All chemicals were used as received.
Diethylaminoethyl cellulose (DEAE-52) anion exchange media was obtained from
Whatman; Sephadex G-50, G-25, and G-15 size exclusion media were obtained from Pharmacia.
Gel electrophoresis reagents, gels and electrophoresis equipment were obtained from Bio-Rad.
The ultrafiltration supplies (8400 and 8010 ultrafiltration units and YC05 membranes) were
obtained from Amicon.
Chicken livers were obtained from local venders; alligator livers were obtained from All
American Gator of Pembroke Park, Florida. Human urine samples were collected from healthy
male volunteers and cooled to 4 °C. Doubly deionized water (dH2O) was used throughout.
2.4
Methods
2.4.1
Chromium Concentration Determination
Chromium concentrations were determined by a modification of the diphenylcarbazide
method of Marczenko using the method of standard additions to minimize matrix effects.7
Samples in 500 µL of 0.05 M NH4OAc, pH 6.5 buffer were examined by adding the reagent
solutions in the following order: 10 µL of 3M NaOH, 30 µL of 30% H2O2, 500 µL of a
diphenylcarbazide solution (0.16 g diphenylcarbazide per 20 mL of ethanol), and 5 µL of
concentrated H2SO4. The absorbance of the solution was measured at 540 nm. For quantitative
assays, a standard solution using CrCl3·6H2O as Cr source was used to generate a standard curve.
2.4.2
Protein and Oligopeptide Concentration Determination
Two methods were used to measure protein and oligopeptide concentrations. After
column chromatography, the concentration of the oligopeptide was determined by a fluorescence
assay that determines the concentration of primary amine. As the N-terminus is the only primary
17
amine in the oligopeptides, the concentration of primary amines is equal to the oligopeptide
concentration. Each sample was diluted to 200 µL, and the following reagents were added to
each sample: 2.00 mL of 0.2 M boric acid at pH 7 and 1.00 mL of fluorescamine solution (0.02 g
fluorescamine per 100 mL acetone). Excitation and emission wavelengths of 390 nm and 475 nm,
respectively, were utilized. A solution of glycine was used as standard. The fluorescamine
solution was made fresh for each assay.8 Total protein content was determined using a Pierce
BCA protein assay kit. Bovine serum albumin was used as a standard. The assay assumes that
proteins have a similar average composition; however, the composition of peptides may deviate
significantly from this. The assay should be considered to have significant error but is adequate
to determine trends.
2.4.3
Isolation of LMWCr from Chicken and Alligator Liver
The purification LMWCr of chicken and alligator liver included homogenization and
centrifugation, ethanol precipitation, anion-exchange chromatography, and gel filtration
chromatography. The procedure followed the procedure described by the Vincent group for the
isolation of LMWCr from bovine liver with a few modifications.1
Fresh liver was diced into 1 inch cubes, and the tissue was suspended and homogenized;
for every 1 kg of tissue; 1 liter of dH2O containing a protease inhibitor cocktail (0.087 g
phenylmethylsulfonyl fluoride, 0.363 g benzamide, and 0.24 mg Pepstatin A) and 0.5 g of
K2Cr2O7 was added. All subsequent operations were performed at 4
. The homogenate was
then centrifuged at 11,000 g for 10 minutes to remove cellular debris. An equal volume of 100%
ethanol was added to the collected supernatant, and the resulting 50% ethanol mixture was
stirred overnight. The supernatant was collected again by centrifugation, and additional 100%
ethanol was added to produce a 90% ethanol mixture. The mixture was allowed to stir for 48 hrs,
18
before being centrifuged at 4200 g for 5 minutes. The solid was collected as a brown powder
after freeze drying. The powder was resuspended with a minimal amount of dH2O and briefly
centrifuged at 4,200 g for 10 minutes to remove any undissolved particles. A filtration through
glass wool was performed to avoid plugging the chromatography column in the next step.
In the following procedures, the Cr content was determined by the diphenylcarbazide
method, and the protein/peptide content was estimated using the absorbance at 260 nm.7 The
clarified greenish-brown filtrate was loaded onto an anion-exchange column of DEAE cellulose
(2.5 × 35 cm), which was equilibrated with 0.05 M NH4OAc buffer, pH 6.5. Next, the column
was washed with at least 3 bed volumes of 0.05 M NH4OAc buffer, pH 6.5. A one liter linear
gradient from 0.05 M, pH 6.5 to 2.0 M NH4OAc buffer, pH 7.2 was applied to the DEAE column.
Fractions containing the Cr-peptide were collected and pooled.
These fractions were
concentrated by ultrafiltration (Amicon 8010 and/or 8400 using YC05 membranes) and diluted
with dH2O before reapplying to an identical anion-exchange column. The washing and elution
procedures were repeated. The Cr-peptide fractions were collected and pooled, concentrated by
ultrafiltration again to less than 7 mL, applied to a Sephadex G-50 size exclusion column (2.5 ×
65 cm), and then eluted with 0.05 M NH4OAc buffer, pH 6.5. The greenish-brown chromiumcontaining fractions were collected and concentrated by ultrafiltration to a volume less than 5 mL.
A Sephadex G-25 column (2.5 × 65 cm) and a Sephadex G-15 column (2.5 × 65 cm) were
utilized successively under similar conditions to further purify the Cr-peptide fractions. The
resulting liver LMWCr solution was concentrated by ultrafiltration and lyophilization. The
product was stored at -20
.
19
2.4.4
Isolation of LMWCr from Human Urine
All procedures were approved by the UA Medical IRB (Institutional Review Board).
Acid solution peptides in urine are relatively resistant to proteinase and peptidase breakdown.9
Ten liters of human urine were collected from one volunteer. As previous attempts to isolate a
Cr-containing species without adding Cr were unsuccessful, 30 mg CrCl3·6H20 was added per
500 mL of urine following Wada and coworkers.10 The product was diluted with an equal
volume of dH2O. A batch extraction was performed using anion-exchange DE-52 media; after
receiving equilibrium, the DE-52 media was isolated by filtration and washed with 3 liters of
dH2O. The media was then poured into a column (5 × 48 cm), and a linear gradient from dH2O
to 2 M NH4OAc buffer, pH 7.2 was applied. After Cr-peptide fractions were collected and
pooled, similar procedures as for the purification of the liver LMWCrs were performed,
including using a second DE-52 column and G-25 and G-15 columns.
2.4.5
Molecular Weight Determination
The molecular weight (MW) of the LMWCr’s was estimated by gel exclusion
chromatography and gel electrophoresis. A Sephadex G-25 column (2.5 × 65 cm) and a
Sephadex G-15 column (2.5 × 65 cm) equilibrated with 0.05 M NH4OAc buffer pH 6.5, were
utilized. Blue dextran was used to determine the void volume of the column, and insulin chain A
(MW = 2532 Da), bradykinin acetate (MW = 1060.21 Da), and glycine (MW = 75.07 Da) were
used as standards. Fractions were assayed for protein by UV spectroscopy at 230 nm.
Gel electrophoresis (sodium dodecyl sulfate polyacrylamide (SDS)), was used to
determine the MW and purity of the LMWCr’s and was performed using a Bio-Rad MiniProtean II electrophoresis cell and precast 16.5% Tris-Tricine Gels, using a Tris-Glycine-HCl
buffer system following a procedure for separation of small peptides.11,12 A mixture of insulin
20
chain A (MW=2531.6 Da) and bradykinin acetate (MW=1060.2 Da) was used as MW standards.
Protein bands were observed by Coomassie blue staining.
2.4.6
Phosphatase Activity Assays
The phosphotyrosine phosphatase, PTP, activity of the LMWCr’s was estimated using p-
nitrophenyl phosphate (p-NPP) by the method of Li et al.13 Phosphatase activity was assayed
using 5 mM p-nitrophenyl phosphate (p-NPP) in 0.05 M Tris, pH 7.5, buffer as the substrate as
previously described.14 Incubation of the chicken material with the substrate proceeded for 1 h at
37 °C. The reaction was terminated by the addition of sodium hydroxide. The hydrolysis of the
phosphate group from p-NPP results in a yellow solution of p-nitrophenoxide with an absorbance
maximum at 404 nm. The amount of chicken material added was determined based on the Cr
concentration of solutions of LMWCr.
2.5
Results and Discussion
2.5.1
Isolation and Purification
LMWCr is thought to exist originally in tissue cytosol as the chromium-free apoLMWCr,
since the formation of chromium-bound LMWCr requires incubation of the tissue homogenate
with Cr.15 Isolation of bovine LMWCr without addition of potassium dichromate in this
laboratory produced a material (used as apoLMWCr in chapter 2) that has approximately the
same amino acid composition (E:D:G = 3.5:2:1.6, cysteine assay was not requested) as the
product with loading using a dichromate salt. This suggests that LMWCr is not an artifact of the
isolation method requiring the involved with Cr(VI) loading.16 Reaction of Cr(VI) with cellular
reductants (such as glutathione, ascorbate, or NAD(P)H-dependent enzymes) ultimately leads to
the formation of stable (kinetically inert) Cr(III) complexes with biological ligands.17,18 An in
vitro chromium loading procedure was utilized to guarantee that the LMWCr contained a full or
21
nearly full complement, allowing the material to be followed during the isolation and purification
by its chromium content. This method has been proved to be effective to increase LMWCr yields
from bovine liver and porcine kidney.1,19
Low-molecular-weight chromium-binding oligopeptides were successfully isolated from
chicken, alligator liver and human urine. Only chromatograms of alligator liver (Figures 2.1 -2.6)
and human urine (Figures 2.7 – 2.11) are presented here. Details for chicken material have been
presented in Michael J.Hatfield’s PhD dissertation.20 The Cr to protein ratio in each purification
procedure reached a constant value at the end of the purification scheme (Table 2.1).
A smaller molecular weight species possessing amino acids and bound Cr formed the
shoulder behind the main band in G-15 elution (Figures 2.5 and 2.10) in both isolation
procedures. The shoulder increased with time suggesting it is a hydrolysis product of LMWCr. A
dimer of urine LMWCr was found in the G-25 elution profile (Figure 2.9 and Table 2.2), which
may be “glued” by cluster of chromic ions (ratio of Cr:peptide ~ 14) caused by adding a large
excess of CrCl3·6H2O.
SDS-PAGE gel electrophosis (Figure 2.12) suggests that the molecular weights of
chicken liver, alligator liver, and human urine LMWCr are all close to 1kDa and that all were
isolated in acceptable purities.
22
2.0
1.8
Protein @ 260 nm
Chromium @ 540 nm
1.6
Absorbance
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
20
40
60
80
100
120
140
Fraction Number
Figure 2.1. Elution profile of alligator liver Cr-containing fractions on first DEAE column.
23
1.4
Protein @ 260 nm
Chrmoium @540 nm
1.2
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
0
20
40
60
80
100
120
140
160
Fraction Number
Figure 2.2. Elution profile of alligator liver Cr-containing fractions on second DEAE
column.
24
1.8
1.6
Protein @ 260 nm
Chromium @ 540 nm
1.4
Absorbance
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
20
40
60
80
100
120
140
Fraction Number
Figure 2.3. Elution profile of alligator liver Cr-containing fractions on Sephadex G-50
column.
25
2.5
protein @260 nm
Chromium @ 540 nm
Absorbance
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
100
120
fraction number
Figure 2.4. Elution profile of alligator liver Cr-containing fractions on Sephadex G-25
column.
26
1.2
Protein @ 260 nm
Chromium @ 540 nm
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0
20
40
60
80
Fraction Number
Figure 2.5. Elution profile of alligator liver Cr-containing fractions on Sephadex G-15
column.
27
0.16
Protein @ 260 nm
Chromium @ 540 nm
0.14
Absorbance
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
10
20
30
40
50
Fraction Number
Figure 2.6. Elution profile of alligator liver Cr-containing fractions on HPLC using a
Shodex OH PAK column.
28
3.5
Protein @ 260 nm
Chromium @ 540 nm
3.0
Absorbance
2.5
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
100
120
140
160
Fraction Number
Figure 2.7. Elution profile of human urine Cr-containing fractions on first DEAE column.
29
3.5
3.0
Protein @ 260 nm
Chromium @ 540 nm
Absorbance
2.5
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
100
120
Fraction number
Figure 2.8. Elution profile of human urine Cr-containing fractions on second DEAE
column.
30
3.5
~2K Da
Protein @ 260 nm
Chromium @ 540 nm
3.0
~1K Da
2
Absorbance
2.5
3
2.0
>5K Da
1.5
1.0
1
0.5
0.0
0
20
40
60
80
Fraction Number
Figure 2.9. Elution profile of human urine Cr-containing fractions on Sephadex G-25
column.
31
2.0
1.8
protein @ 260 nm
Chromium @ 540 nm
1.6
Absorbance
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Fraction Number
Figure 2.10. Elution profile of human urine Cr-containing fractions on Sephadex G-15
column.
32
0.04
Protein @ 260 nm
Chromium @ 540 nm
Absorbance
0.03
0.02
0.01
0.00
0
10
20
30
40
50
60
Fraction Number
Figure 2.11. Elution profile of human urine Cr-containing fractions on HPLC using a
Shodex OH PAK column.
33
Table22.1. Purification of LMWCr from chicken, alligator liver and human urine
Purification step
Chicken (13 kg liver)
mg Cr
mg protein
mg Cr/mg protein
Homogenate
1st DEAE
2nd DEAE
G50
G25
G15
Alligator (2.5 kg liver)
101
41.2
17.5
11.9
3.70
2.50
1.19 x 104
2.17 x 103
675
156
42.6
32.5
0.00850
0.0150
0.0260
0.0763
0.0869
0.0770
Homogenate
1st DEAE
2nd DEAE
G50
G25
G15
Urine (10 liter)
6.13
3.60
3.51
3.10
1.02
0.444
996
837
392
27.7
22.9
12.1
0.00615
0.00430
0.00895
0.1119
0.0445
0.0367
1st DEAE
2nd DEAE
G25
G15
37.4
18.0
2.1
1.32
167.4
133.9
25.1
16.7
0.2234
0.1344
0.0837
0.0792
34
Figure 2.12. SDS-PAGE gel electrophoresis of chicken, alligator and human urine LMWCr.
35
2.5.2
Physical Characterization
The isolated LMWCr's from chicken and alligator liver have a Cr/peptide ratio of
2(± 0.3):1. This number for both materials is about one-half the ~3.5:1 ratio found in isolated
bovine LMWCr. A higher ratio, 5:1, was found for human urine LMWCr. Amino acid analysis
indicates that chicken and alligator LMWCr are composed primarily of aspartate, glutamate,
glycine, cysteine, and serine in an approximate ratio of 3:5:2:2:1, while urine has an approximate
composition of 4:4:1:1:0 (Table 2). The amino acid composition is very similar to LMWCr's
from other sources. Efforts to sequence the oligopeptides using Edman degradation have been
unsuccessful. No confirmed mass spectral signal has been acquired for the chicken, alligator and
human urine LMWCr by MALDI and ESI ionization methods. This is not uncommon for Crcontaining acidic peptides.21 More about sequencing by MS will be discussed in chapter 4.
2.5.3
Electronic Spectroscopic studies
The ultraviolet spectra of mammalian LMWCr's reported to date contain a broad band or
shoulder at ~260 nm.1,19,22,23 The ultraviolet spectra of LMWCr's from chicken liver and alligator
liver also possess this feature (Figures 13 and 14). The spectrum for human urine LMWCr does
shift to ~320 nm (Figure 2.15). This feature may arise from a disulphide linkage, consistent with
the presence of 2 cysteine residues in the oligopeptides.17 The amino acids aspartate, glutamate,
cysteine and glycine do not show any absorbance at 260 nm except for cysteine when oxidized to
cystine. The spectra in the visible region are dominated by two maxima at 397 (ε/Cr = 434 M− 1
cm− 1) and 578 (ε/Cr = 214 M− 1 cm− 1) for the chicken liver material. These bands are shifted to
410 and 563 nm for the alligator liver material and to 394 and 571 nm for urine material. These
ligand field and are assigned to 4A2g→4T2g (
1)
and 4A2g→4T1g
(
2)
spin-allowed dÆd
transitions, respectively. The energy of the first transition is equal to the crystal field parameter
36
37
Table22.2. Comparison of the amino acid cmposition of LMWCr’s and the Cr:peptide ratios from different sources
10Dq of the d3 Cr(III). The Racah parameter B was calculated using the formula:
1 2
3
9
5
with ν1 and ν2; the values reflect all or predominantly oxygen coordination and are similar to
those reported for bovine liver LMWCr (Table 3).1 For d3 octahedral complexes, three spinallowed transitions are predicted at energies. ν1 and ν2 always can be observed in visible region
while ν3 falls in to ultraviolet area. Here, ν3 was calculated by formula ν
225B
100Dq
7.5B
15Dq
180BDq ; the values of ν3 of the LMWCr’s are near 253 nm, which
could be another contribution to 260 nm absorbance. Additionally, weak absorptions in the
region of the spectra between 680 and 700 nm may be assigned to spin-flip transitions. Also
present are some very weak features between 300 and 400 nm, which for bovine liver LMWCr
have tentatively been assigned to double excitation transitions, suggesting the presence of a
multinuclear assembly in chicken and alligator LMWCr.
2.5.4
Electron Paramagnetic Resonance
Additional evidence for structural similarities among the multinuclear Cr(III) assemblies
of the LMWCr's from animal livers and human urine comes from EPR spectroscopy. The Xband EPR spectra collected at 10 K of the chicken and alligator LMWCr's display a broad signal
at g ~ 1.97 and smaller features around g = 5 (Figure 2.16); the spectra are nearly identical to that
of bovine LMWCr.24 For bovine liver LMWCr, the signals were shown to arise from ground spin
states. The broad signal at g ~ 2 arises from a S = 1/2 state; given that Cr(III) has a spin of 3/2,
the signal is presumed to arise from the antiferromagnetic coupling of three chromic centers to
give an assembly with an overall spin of S = 1/2. The fourth chromic center (S = 3/2) then rests
in a mononuclear site and gives rise to the signals at g ~ 5. The signal at g ~ 5 is more intense for
the alligator spectrum than the chicken spectrum. Thus, this site would appear not to be fully
38
[a]
[b]
Figure 2.13. (a) Ultraviolet spectrum of chicken liver LMWCr in 50 mM ammonium
acetate, pH 6.5. [Cr] = 0.2 mM; (b) visible spectrum of chicken liver LMWCr in 50 mM
ammonium acetate, pH 6.5. [Cr] = 1.1 mM.
39
[a]
2.0
Absorbance
1.5
1.0
0.5
240
260
280
300
320
340
360
380
400
Wavelength (nm)
[b]
Absorbance
3
2
1
0
300
400
500
600
700
800
Wavelength (nm)
Figure 2.14. [a] Ultraviolet spectrum of alligator liver LMWCr in 50 mM ammonium
acetate, pH 6.5. [Cr] = 0.6 mM; Visible spectrum of alligator liver LMWCr in 50 mM
ammonium acetate, pH 6.5. [Cr] = 4.7 mM.
40
[a]
Absorbance
3
2
1
0
240
260
280
300
320
340
360
380
400
Wavelength (nm)
[b]
2.0
Absorbance
1.5
1.0
0.5
0.0
400
500
600
700
800
Wavelength (nm)
Figure 2.15. [a] Ultraviolet spectrum of human urine LMWCr in 50 mM ammonium
acetate, pH 6.5. [Cr] = 0.5 mM; [b] Visible spectrum of alligator liver LMWCr in 50 mM
ammonium acetate, pH 6.5, [Cr] = 2.8 mM.
41
Table 2.3. Comparison of visible spectra of bovine, chicken, alligator and human urine
LMWCr
Bovine
Chicken
Aligator
LMWCr
LMWCr
LMWCr
ν1
394 nm
397 nm
406 nm
394 nm
ν2
576 nm
578 nm
565 nm
571 nm
Dq
1736 cm-1
1730 cm-1
1770 cm-1
1751 cm-1
B
851 cm-1
832 cm-1
688 cm-1
824 cm-1
ν3(calculated) 253 nm
253 nm
257 nm
253 nm
Spin-flip
~690 nm
~690 nm
—
~690 nm
transition
42
Urine LWMCr
loaded with chromium in the chicken and alligator LMWCr's and could account for the low
Cr/oligopeptide ratio. The broadening of the S = 1/2 signal from the bovine material is believed
to arise from a dipolar relaxation pathway resulting from interactions with the mononuclear
center.24 The proposed arrangement of the chromium(III) centers in bovine liver LMWCr was
also supported by the results of variable temperature magnetic susceptibility and X-ray
absorption studies.24 Thus, the similar EPR spectra for all three liver LMWCr's suggest that all
possess similar trinuclear and mononuclear arrangements of Cr(III) centers. Chromic ions bind
with bovine apoLMWCr in a highly cooperative fashion with a Hill coefficient of 3.47, which
suggests that it only exists in either the fully loaded holo form or the apo form of the
oligopeptide.25 Thus, only half of the chicken material may exist in the holo form with the other
half in the apo form, and the observed EPR spectrum may only be due to oligopeptide with four
chromic ions.
Human urine LMWCr gave an X-band EPR spectrum (Figure 2.17) having a sharp
feature at g = 4, which is very different from that of chicken and alligator LMWCr’s. The g
value of ~ 4 is consist with the presence of a mononuclear S =3/2 Cr(III) center.24 Chromiumcarbon σ bonds complex with effective g value g ~ 4 and 2 are typical of an electronic spin S=3/2
with moderately large zero field splitting (ZFS) and small rhombicity (D>hν = 0.3 cm-1 at Xband; E/D = 0).26 While MS studies suggest Cr(III) can coordinate with sites on the peptide
backbone, this is extremely unlikely to occur in solution.21 However, highly aquated Cr(III)
species might interact weakly with the anionic peptide. The sharp g = 4 signal may caused by
multiple Cr’s interacting weakly with the peptide; this could contribute to the high Cr:peptide
ratio of human urine LMWCr (average >7 for bands 2 and 3 of the G25 column).27,28
43
2
X-ban
nd EPR speectrum of chicken livver and alliigator LMW
WCr in 500 mM
Figure 2.16.
ammonium acetatee buffer, pH
p 6.5. (T
Top) Chickeen liver LMWCr.
L
[C
Cr] =
15 mM.
Instrumental param
meters: mod
dulation freq
quency, 1000 kHz; modu
ulation amp
plitude, 10.559 G;
time con
nstant, 20.48 ms; sweep
p time, 83.89 s; field center,
c
40000 G; sweep width, 76000 G;
frequenccy, 9.6327 GHz; and
d power, 2.0
2
mW. (Bottom) Alligator liver LMW
WCr.
[Cr] = ~115 mM. Insstrumental parameterrs: modulattion frequeency, 100 kH
Hz; modulaation
amplitud
de, 10.59 G;
G time constant, 20.488 ms; sweep
p time, 83.89 s; field center, 40000 G;
sweep width, 7600 G;
G frequenccy, 9.6308 GHz;
G
and poower, 2.0 mW
W.
44
g = 4.0
g = 2.0
3000
EPR signal intensity
2800
2600
g = 4.7
2400
2200
2000
1800
1600
1000
2000
3000
4000
5000
Magnetic Field / Gauss
Figure 2.17. X-band EPR spectrum of urine LMWCr in 50 mM ammonium acetate buffer,
pH 6.5. [Cr] = ~13.5 mM. T = 77 K, Instrumental parameters: Modulation amplitude 5.0 G;
frequency, 9.047 GHz and power, 2.0 mW. time constant = 0.5 s; gain = 5 x 104; 2 scans.
45
2.5.5
Biological activity
As bovine liver LMWCr has been shown to activate an adipocyte membrane-associated
phosphotyrosine phosphatase and the tyrosine kinase activity of insulin receptor, similar
activities were probed for the chicken and alligator liver LMWCr's. Before testing whether the
alligator or chicken materials could activate membrane phosphatase activity, they were examined
for any inherent phosphatase activity. Both materials (Cr concentration 10 and 100 μM) were
able to catalyze the hydrolysis of p-nitrophenyl phosphate; this activity was dependent on the
concentration of LMWCr and could not be separated from the material by additional
chromatography. The results were consistent for each purification of the chicken and alligator
liver materials; the specific activities were 2.17 × 10− 4 and 1.02 × 10− 3 mol p-nitrophenol/mg
oligopeptide/min for the alligator and chicken materials, respectively. Thus, these avian and
reptilian materials may have an inherent phosphatase activity, although care needs to be taken in
interpreting these results as the presence of persistent traces of phosphatase enzymes in isolated
proteins or oligopeptides is not an uncommon complication. When the effects of a range of
concentrations of the LMWCr's on adipocytic membrane phosphatase activity were examined,
the phosphatase activity was never greater than the sum of the inherent activity of the
membranes plus the activity of the LMWCr's. Kinase activity assays were not attempted because
of the complications arising from the phosphatase activity.
2.6
Conclusions
Low-molecular-weight chromium-binding substance (LMWCr) has been isolated from
the liver of aves and reptiles, as well as human urine, in addition to the liver of mammals. All
LMWCr's are similar in amino acid composition and molar mass but not identical. EPR and
electronic spectroscopic studies suggest all LMWCr’s possess structurally similar multinuclear
46
Cr(III) assemblies with the Cr(III) centers in a pseudo-octahedral environment of oxygen-based
ligands.
This suggests that LMWCr may play an important role in these organisms, including
potentially a role in the insulin-signaling pathway as proposed for mammalian LMWCr. The
discovery of similar analogs in animal tissue and human urine provide some base for purposed
mechanism of LMWCr release into the bloobstream and ultimately urine after binding insulin
receptor. The results of the investigations described herein should be catalysts to further studies
exploring the biochemical role of chromium.
47
REFERENCES
(1)
Davis, C. M.; Vincent, J. B. Archives of Biochemistry and Biophysics 1997, 339, 335-343.
(2)
Lance, V. A.; Elsey, R. M.; Coulson, R. A. General and Comparative Endocrinology
1993, 89, 267-275.
(3)
Dupont, J.; Chen, J. W.; Derouet, M.; Simon, J.; Leclercq, B.; Tais, M. Journal of
Nutrition 1999, 129, 1937-1944.
(4)
Vincent, J. B. Journal of Nutrition 2000, 130, 715-718.
(5)
Reichelt, K. L.; Johansen, J. H.; Titlestad, K.; Edminson, P. D. Biochemical and
Biophysical Research Communications 1984, 122, 103-110.
(6)
Strong, K. J.; Osicka, T. M.; Comper, W. D. Journal of Laboratory and Clinical
Medicine 2005, 145, 239-246.
(7)
Marczenko, Z. Separation and spectrophotometric determination of elements, 1986.
(8)
Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science
(Washington D C) 1972, 178, 871-872.
(9)
Noguchi, T.; Okiyama, A.; Naito, H.; Kaneko, K.; Koike, G. Agricultural and Biological
Chemistry 1982, 46, 2821-2828.
(10)
Wu, G. Y.; Wada, O. Japanese Journal of Industrial Health 1981, 23, 505-512.
(11)
Lutz, M. P.; Pinon, D. I.; Miller, L. J. Analytical Biochemistry 1994, 220, 268-274.
48
(12)
Sarfo, K.; Moorhead, G. B. G.; Turner, R. J. Letters in Peptide Science 2003, 10, 127-133.
(13)
Li, J. P.; Elberg, G.; Gefel, D.; Shechter, Y. Biochemistry 1995, 34, 6218-6225.
(14)
Davis, C. M.; Sumrall, K. H.; Vincent, J. B. Biochemistry 1996, 35, 12963-12969.
(15)
Yamamoto, A.; Wada, O.; Ono, T. Journal of Inorganic Biochemistry 1984, 22, 91-102.
(16)
Levina, A.; Harris, H. H.; Lay, P. A. Journal of the American Chemical Society 2007,
129, 9832-9832.
(17)
Levina, A.; Codd, R.; Dillon, C. T.; Lay, P. A. Progress in Inorganic Chemistry, Vol 51
2003, 51, 145-250.
(18)
Connett, P. H.; Wetterhahn, K. E. In Inorganic Elements in Biochemistry Berlin, 1983;
Vol. 54, p 93-124.
(19)
Sumrall, K. H.; Vincent, J. B. Polyhedron 1997, 16, 4171-4177.
(20)
Hatfield, J. M., The University of Alabama, 2005.
(21)
Pu, D.; Vincent, J. B.; Carolyn, J. C. Journal of Mass Spectrometry 2008, 43, 773-781.
(22)
Yamamoto, A.; Wada, O.; Ono, T. European Journal of Biochemistry 1987, 165, 627631.
(23)
Yamamoto, A.; Wada, O.; Suzuki, H. Journal of Nutrition 1988, 118, 39-45.
(24)
Jacquamet, L.; Sun, Y. J.; Hatfield, J.; Gu, W. W.; Cramer, S. P.; Crowder, M. W.;
Lorigan, G. A.; Vincent, J. B.; Latour, J. M. Journal of the American Chemical Society
2003, 125, 774-780.
(25)
Sun, Y. J.; Ramirez, J.; Woski, S. A.; Vincent, J. B. Journal of Biological Inorganic
Chemistry 2000, 5, 129-136.
49
(26)
Mabbs, F. E.; Collison, D. Electron Paramagnetic Resonance of d Transition Metal
Compounds Elsevier: Amsterdam, 1992.
(27)
Shaham, N.; Cohen, H.; Meyerstein, D.; Bill, E. Journal of the Chemical Society-Dalton
Transactions 2000, 3082-3085.
(28)
Keech, A. M.; LeBrun, N. E.; Wilson, M. T.; Andrews, S. C.; Moore, G. R.; Thomson, A.
J. Journal of Biological Chemistry 1997, 272, 422-429.
50
3.
CHAPTER 3
THE BINDING OF TRIVALENT CHROMIUM TO CANDIDATE SEQUENCES OF
LOW-MOLECULAR-WEIGHT CHROMIUM-SUBSTANCE (LMWCr)
3.1
Introduction
To date at least one metallobiomolecule has been well characterized in regards to its
function and mode of action for each essential transition element (the first row metal from
vanadium to zinc, molybdenum, and tungsten) except chromium. After more than five decades of
investigation, no such biomolecule has unambiguously been characterized for chromium. Two
biomolecules are known to bind Cr in vivo: transferrin and low-molecular weight chromiumbinding substance (LMWCr), also termed chromodulin. Transferrin appears to be responsible for
maintaining chromium supplies in the bloodstream and transporting Cr to the tissues and
possibly from the intestine lining to the bloodstream.1,2 LMWCr has been proposed to keep the
insulin receptor’s (IR) active conformation from reversing by binding to the tyrosine kinase
active site of the IR in insulin-dependent cells. These cells are capable of responding to insulin
signaling in response to high blood glucose levels, which are a problem for patients with adultonset diabetes.3 The amino acid composition of peptide of bovine LMWCr is reportedly
E:G:C:D::4:2:2:2, though its sequence has not been obtained.10 Sequencing with Edman
degradation failed as a possible result of bound Cr, an organic modification in vivo or during
sequencing (such as the formation of pyroglutamate when a new glutamate residue is exposed).
51
The use of mass spectrometry to sequence the peptide has proved challenging. Two
ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption
ionization (MALDI), were tested. Biological LMWCr samples, after final purification by
Sephadex G-15 column chromatography, generate only weak signals by MALDI, which is more
tolerant of impurities than ESI. The molecular weight of bovine LMWCr is believed to be 1438
Da, which was confirmed under both positive and negative modes.4 The post-source decay (PSD)
spectrum previously acquired could not be interpreted because of the presence of Cr which
resulted in altered mechanisms of fragmentation.5
By searching the human genome for peptide fragments containing the amino acid
composition
of
bovine
LMWCr,
two
decapeptide
sequences,
EDGEECDCGE
and
DGEECDCGEE, were identified, both in the disintegrin domain of ADAM19 (A Disintegrin and
Metalloproteinase domain 19; GeneID: 8728).6 ADAM19 contains a metalloprotease, disintegrin,
a cysteine-rich, epidermal growth-factor-like, transmembrane, and acytoplasmic domains.7
Specifically, the protein contains a HexxHxxxxxH zinc metalloproteinase moiety and is believed
to be an active protease.8,9 The location of the codons for these two sequences is at the very end
of an exon, suggesting the possibility that LMWCr could be a product derived from alternative
splicing.6 A peptide with the sequence pEEEEGDD or pEEEGDD (where pE is pyroglutamate)
was obtained during attempts to remove the Cr from LMWCr using trifluoroacetic acid (TFA).
The generation and characterization of this heptapeptide is discussed in chapter 4.
Despite its small size, the apoLMWCr isolated from rabbit and bovine liver tightly binds
four equivalents of chromic ions with a binding constant on the order of 1021 M-4 at physiological
pH in vitro. The binding is highly cooperative (Hill coefficient, n=3.47) such that only two
species Cr4-LMWCr and apoLMWCr coexist in solution and apoLMWCr can accept chromic
52
ions from biological molecules including Cr2-transferrin.2 Difference UV spectral studies
corroborated these findings.57
This chapter is designed to study the binding of chromic ions to synthetic decapeptides
and heptapeptides that are potential candidates for the sequence of LMWCr or may comprise part
of the organic portion of LMWCr . The properties of the Cr complexes of these peptides were
compare to those reported for LMWCr from bovine liver.
3.2
Instrumentation
Gamma counting was performed on a Packard Cobra II auto-gamma counter. Atomic
absorption analysis was processed with a Perkin Elmer AAnalyst 400 with HGA 900 graphite
furnace.
All
ultraviolet/visible
HewlettPackard
8453
or
spectroscopic
Beckman
Coulter
measurements
DU800
were
recorded
spectrophotometer;
with
a
fluorescence
measurements were collected with a FluoroMax-3 spectrofluorimeter from Jobin Yvon Horiba.
pH was measured by using a glass electrode (Denver Instrument pH/ATC #300729.1).
3.3
Materials
3.3.1
Synthetic Peptides
ApoLMWCr was isolated in Dr. Vincent’s group at The University of Alabama using a
published method with some modification.10 The peptide EDGEECDCGE was purchased from
Synpep Corp. Peptides; the two sequences DGEECDCGEE and pEEEEGDD were synthesized
by the Cassady group of The University of Alabama. The purities of all the synthetic peptides
were checked by HPLC (High-performance liquid chromatography) using a Shodex OH PAK
B803 column (Shoko Co. Ltd).
3.3.2
Other Chemicals
53
51
CrCl3 was obtained from ICN; CrCl3 was obtained from Fisher Scientific. Hepes was
obtained from Research Organics, Inc.; chromium standard solution was obtained from Perkin
Elmer. Solutions of 0.01 M NaOH and HCl were used for pH adjustment.
3.4
Methods
3.4.1
Physical Characterization of Cr-peptide Complex Studies
Ten equivalents of Cr, in the form of an aqueous solution of chromic chloride
hexahydrate were added to the synthetic peptides in 1 mL of 0.1 M Hepes buffer, pH 7.4; the
mixture was allowed to shake overnight at 4 °C. The solution was loaded onto a Sephadex G-10
column (2.5 × 40 cm) to separate any unreacted Cr from the oligopeptide. The Cr concentration
of the Cr-peptide complex was determined by atomic absorption spectrometry using a graphite
furnace atomizer. A Cr standard solution from Perkin Elmer was used to generate standard
solutions. The temperature program is stated in Table 1.11
The concentration of the oligopeptide was determined by a fluorescence assay that has
been described in Chapter 2. Electronic spectra of each Cr-peptide complex were acquired from
200 to 800 nm. For the ultraviolet spectral change studies, a 1.0-mL solution of synthetic peptide
in 0.1 M, pH 7.4 Hepes buffer with different initial concentrations of CrCl3 was made. The
mixtures were stirred overnight at 4 °C. As REFERENCES, aliquots of chromic ion solution and
buffer were added to the same volume of buffer.
3.4.2
ApoLMWCr and Synthetic Peptides Studies
Studies were carried out by adding aliquots of a mixture of CrCl3 and 51CrCl3 to generate
different concentrations of Cr(III) while maintaining the synthetic peptide in solution at a
constant volume. Known amounts (about 4.6 x 10-7 moles) of peptide and 200 ml of 0.1 M Hepes
buffer (pH 7.4) were slowly stirred in an Amicon 8400 ultrafiltration unit (with a YC05
54
membrane) at 4 °C temperature for at least 12 h to achieve equilibration. The ultrafiltration unit
was then pressurized, and effluent was collected. The content of free chromic ion in the effluent
was determined by gamma counting (Scheme 3.1).
3.4.3
Miscellaneous
Chromium binding experiments were performed in triplicate, and the synthetic peptide
used in at least one of the three sets of triplicates was from a different synthesis. Errors are
presented throughout as standard deviations of the triplicate analyses. Doubly deionized water
(ddH2O) was used throughout.
3.5
Results and Discussion
If a synthetic peptides has a composition and sequence similar to that of apoLMWCr, its
binding constants for chromic ions should be close to those of apoLMWCr. This was probed in
two manners. First, the binding of chromic ion to the candidate peptides was examined. Also, the
UV/Vis spectra of the Cr-peptide complexes were compared.
Net charges of the decapeptides and heptapeptide are -7 and -6.1, respectively, when the
pH of solution is 7.4. Ion exchange between Cr and protons in solution should be taken into
account. Both peptides are rich in carboxylate groups that can bind extra Cr(III) ions on its
surface in addition to reactive sites.12 Cr(III) may coordinate to the donor nitrogen atom of the Nterminal amine and oxygen donor atoms of the carboxylate groups. As a result, ion-exchange,
complexation and adsorption to the surface should be expected with the increase of chromic ions
in solution.
3.5.1
Quantitative Analysis of Cr Bound to Peptides
A mixture of synthetic peptide and 10 equivalents of CrCl3·6H2O was concentrated by
lyophilization and applied to a Sephadex G10 column (2.5 × 40 cm). The Cr-peptide eluted near
55
Table 3.1. Temperature program of atomic adsorption assay for Cr(III) in solution
Step
Temp (°C)
Ramp
Time Hold
Time Gas flow
Gas type
(s)
(s)
(mL min-1)
110
1
20
250
100% Ar
130
15
20
250
100% Ar
1250
30
15
250
100% Ar
1250
1
5
250
100% Ar
atomization 2300
0
5
0
100% Ar
cleanout
1
5
250
100% Ar
Drying
Ashing
2500
56
read
read
Scheme 3.1. Flow chart for study of Cr(III) binding by peptides.
57
void volume of the column. The band with a major absorbance at 260 nm in the elution profile
was collected (Figure 3.1). The Cr:peptide ratio was 2.0(±0.3) for both of decapeptides,
EDGEECDCGE and DGEECDCGEE. In agreement, no m/z peak corresponding to more than 2
Cr bound to the peptides has been observed in MALDI MS spectra for these two peptides.13 For
the peptide pEEEEGDD, the ratio of Cr bound to peptide cannot be determined because the
fluorescamine assay does not work for a cyclic amide at the N-terminus.
Difference ultraviolet spectra were also collected during the titration of the synthetic
peptides with chromic ions. The additional of the first few equivalents of chromium to the
synthetic peptide has little effect on the spectra (Figures 3.2 and 3.3). However, the addition of
excess chromium results in a quick increase of an intense ultraviolet absorption feature.
3.5.2
Effect of Cr(III) concentration
A variation of the equilibrium dialysis method using an ultrafiltration device was utilized
to examine the binding of chromium to the synthetic peptides to determine their ability to bind
chromium.
The effect of Cr(III) concentration on the binding by the peptides was investigated by
gradually increasing Cr(III) concentration in solution at pH of 7.4 at 4 °C. Hepes buffer kept
solution pH around 7.4. The binding study was carried out for 12 h to ensure the equilibrium was
attained.
The results (Figure 3.4) reveal that metal uptake is increased with increases in metal ion
concentration. This can be explained by a progressive increase in electrostatic interaction,
relative to covalent interaction (vide infra).12,14 The results presented here indicate that all the
synthetic peptides showed an ability to absorb chromic ions from aqueous solution as
apoLMWCr does.
58
0.7
0.6
Absrobance
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
60
80
Fraction number
Figure 3.1. Elution profile of Cr-oligopeptide applied to Sephadex G-10 column (260 nm).
59
1:1
2:1
4:1
10:1
Absorbance
0.6
0.4
0.2
0.0
200
250
300
350
400
Wavelength
Figure 3.2. UV spectra of species formed upon the addition of varying quantities of chromic
ions to the peptide EDGEECDCGE. Numbers in inset correspond to Cr:peptide ratios.
[peptide] = 0.18 mM.
60
0.40
1:1
2:1
4:1
10:1
0.35
Absorbance
0.30
0.25
0.20
0.15
0.10
200
250
300
350
400
Wavelength (nm)
Figure 3.3. UV spectra of species formed upon the addition of varying quantities chromic
ions to peptide DGEECDCGED. Numbers in inset correspond to Cr:peptide ratios.
[peptide] = 0.18 mM.
61
8
apoLMWCr
EDGEECDCGE
DGEECDCGEE
pEEEEGDD
qM(mmol/g peptide)
6
4
2
0
0
2
4
6
8
10
12
14
Ratio of [Cr]:[peptide]
Figure 3.4. Sorption isotherm of Cr(III) ion on apoLMWCr and synthetic peptides. Volume
of adsorption medium: 200 mL, temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4
62
Adsorption data for a wide range of adsorbate concentrations are most accurately
described by adsorption isotherms, such as those of Langmuir or Freundlich, which relate
adsorption amount to the equilibrium adsorbate concentration in the bulk fluid phase. The simple
Langmuir model assumes a uniform distribution of energetic adsorption sites, a 1:1
stoichiometry whereby one metal ion binds to one binding site.15
For simplicity’s sake, binding models considered here involved only one type of binding
site and one type of metal ion. The reaction for the binding of protons H to binding site B is
B
H
BH . The corresponding equilibrium expression is
eqn (1)
where KH is the binding or formation constant of protons and [H] is the concentration of protons
in solution. The reaction for the binding of metal ion M to binding site is
.
The corresponding equilibrium expression is
eqn (2)
where KM is the binding or formation constant of metal ions and [M] is the concentration of
metal ions in solution, which is chromic ions in this case. The total number of binding sites is
given by
eqn (3)
For this model, an explicit pH-sensitive isotherm equation is needed to calculate the metal
binding as a function of metal concentration, and the pH can be derived from equations (1) to (3).
The amount of metal bound to peptide is qM (mmol/g peptide)
eqn (4)
63
where Bt is total number of binding sites. For constant pH,
eqn (5)
which when substituted into equation (4) yields
eqn (6)
Rearranging then gives
eqn (7)
This can be rearranged to give the Langmuir adsorption isotherm equation (equation (8)), which
has a form that can readily be plotted and
eqn (8)
where K = Langmuir equilibrium constant, [M] = aqueous chromic ion concentration, qM =
amount adsorbed, and Bt = maximum amount adsorbed as [Cr] increases.
The degree of linearity of the data indicates how appropriate the model is for the data.
Although the Langmuir isotherm is generally regarded as empirical, some attempts have been
made to derive some information about binding trends from the empirical Langmuir curve.
Figure 3.5, however, shows that Cr binding to apoLMWCr can be represented by two
intersecting straight lines with different slopes. The chromium:oligopeptide ratio at the
intersection point is 3.6, which is very close to the average amount of chromium bound to
isolated bovine LMWCr, which is 3.5.10 Similar intersection points were found when using the
Langmuir process for examining data on the other synthetic peptides; the points occurred at
Cr:peptide ratios of 2, 2, 4 for EDGEECDCGE, DGEECDCGEE and pEEEEGDD, respectively
(Figures 3.6-3.8). This is consistent with the Cr:peptides binding ratios found after
64
6
5
1/qM (mmol/g)
-1
4
3
[Cr]:[peptide]=3.6
2
1
0
0
200
400
600
800
1/[M] (mM)-1
Figure 3.5. Langmuir linear isotherm of Cr(III) adsorbed on apoLMWCr, Volume of
adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4.
65
25
1/qM (mmol/g)
-1
20
15
10
[Cr]:[peptide]=2
5
0
0
2000
4000
6000
8000
10000
1/[M] (mM)-1
Figure 3.6. Langmuir linear isotherm of Cr(III) adsorbed on EDGEECDCGE, Volume of
adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4.
66
18
16
14
1/qM (mmol/g)
-1
12
10
8
[Cr]:[peptide]=2
6
4
2
0
-2
0
1000
2000
3000
4000
5000
1/[M] (mM)-1
Figure 3.7. Langmuir linear isotherm of Cr(III) adsorbed on DGEECDCGEE, Volume of
adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4.
67
5
1/qM (mmol/g)
-1
4
3
2
[Cr]:[peptide]=4
1
0
0
100
200
300
400
500
600
1/[M] (mM)-1
Figure 3.8. Langmuir linear isotherm of Cr(III) adsorbed on pEEEEGDD, Volume of
adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial pH: 7.4.
68
chromatography
(apoLMWCr:
3.6
vs.
3.5,
EDGEECDCGE:
2
vs.
2.0(±0.3),
and
DGEECDCGEE: 2 vs. 2.0(±0.3)), which maysuggest when enough chromic ions are present in
solution to saturate all of binding sites on peptide and that excess chromic ions continue to “bind”
on the peptide but in a different mode(s). A change in mode from specific coordinate covalent
binding to just electrostatic absorption on the peptide surface is proposed. Furthermore, the
inflection point of peptide pEEEEGDD implies that it may bind up to four chromic ions.
To further compare the binding properties between the synthetic peptides and
apoLMWCr in CrCl3 solution, the method of Hill was applied to measure of the degree of
cooperativity between the covalent binding sites with the initial low amounts of Cr(III) in
solution, assuming covalent binding sites are occupied before surface adsorption occurs.16 After
the total number of binding sites was determined using the Langmuir isotherm, the method of
Hill was utilized. This method uses the average binding number y defined as
eqn (9)
where Kf is the binding of formation constant and n is the Hill constant such that
eqn (10)
Hill plots gave linear curves (Figures 3.9- 3.12). Kf and n were obtained as the value of yintercept and slope, respectively (Table 2.2). These data indicate a large degree of positive
cooperativity such that the binding of the first and subsequent chromic ions facilitates the
binding of addition Cr, perhaps in a multinuclear assembly; the magnitudes suggest that
essentially only peptide or peptide saturated with chromic ions exist in solution.
Hill constants, Kf and n, of apoLMWCr differ only slightly from published data, Kf =
1.54 x 1021 M-4 and n = 3.47. Hill constants for EDGEECDCGE and pEEEEGDD are closer to
that of apoLMWCr than to that of DGEECDCGEE. For EDGEECDCGE, the Hill constant is
69
greater than the number of interacting sites, as only two Cr(III) binding sites are on the peptide.
This suggests that the resulting Cr-peptide complex is actually a dimer of peptide.17
Negative values of qM and K (Table 3) were unexpected using the Langmuir linear
isotherm (Figure 3.13). The appearance of negative Bt values for all peptide suggests the
limitation of using the simple Langmuir model on cases of tight-binding. The negative value of
Bt indicates that most of the sorption sites have a high affinity for chromic ion, especially at low
concentration (more high negative y-intercept than that at high concentration).18 Models of
multimetal, multisite biosorption were also considered as trivalent of Cr(III) is known to form
multinuclear complexes with short peptides.19
Using a model based on a 1:2 or other binding stoichiometries whereby one chromic ion
may bind to two or more binding sites may be more appropriate. Bidentate formation of B2M or
BM0.5 was tested. Modified equations,
√
eqn (11)
and
√
eqn (12)
were used to plot data for each peptide .20 A closer approach to linearity was obtained for BM0.5
model for apoLMWCr and EDGEECDCGE (Figures 3.14 and 3.15), but inflection points and
negative Y-intercepts (if all points were utilized) were still generated. Thus, the use of more
complicated Langmuir isotherm models did not significantly the results.
3.5.3
UV/Vis spectra of the Cr(III) loaded Synthetic Peptide
The electronic spectra of the Cr-peptide complexes of EDGEECDCGD and
DGEECDCGDE both feature one maxima in the ultraviolet region at 287 nm (Figures 3.16 and
70
17). These spectra are not similar to that of LMWCr, which has an obvious absorbance shoulder
at 260 nm.10
pEEEEGDD has absorbance maxima or shoulders in both the visible and ultraviolet
regions, which are at ~270 nm (shoulder), 411 nm and 577 nm (Figure 3.18). Their intensity and
position both are similar to those of a bovine LMWCr and are characteristic of a chromium(III)
center in an octahedral environment of oxygen-based ligand.10 The reason for absorbance shift
still needs further investigation. Also ultraviolet absorbance of Cr-pEEEEGDD at ~270 nm
brings into question whether the ultraviolet absorbance shoulder at 260 nm in LMWCr is caused
by disulfide bond, as has previously been proposed.21
71
1.5
1.0
log[y/(1-y)]
0.5
0.0
-0.5
-1.0
-1.5
-5.8
-5.6
log [Cr]
Figure 3.9. Hill plot of chromic ion binding to apoLMWCr.
72
-5.4
-5.2
1.0
0.5
log[y/(1-y)]
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-7.0
-6.8
-6.6
-6.4
log[Cr]
Figure 3.10. Hill plot of chromic ion binding to peptide EDGEECDCGE.
73
-6.2
1.5
1.0
log[y/(1-y)]
0.5
0.0
-0.5
-1.0
-1.5
-6.8
-6.6
-6.4
-6.2
-6.0
-5.8
log[Cr]
Figure 3.11. Hill plot of chromic ion binding to peptide DGEECDCGEE.
74
-5.6
-5.4
0.6
0.4
0.2
log[y/(1-y)]
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-5.7
-5.6
-5.5
-5.4
-5.3
log[Cr]
Figure 3.12. Hill plot of chromic ion binding to synthetic peptide pEEEEGDD.
75
-5.2
Table33.2. Hill plot constants of apoLMWCr and synthetic peptides.
Kf
n
R2
Qualititive
ApoLMWCr
1.10 x 1021 M-4
(bovine liver)
EDGEECDCGE 1.48 x1023 M-2
3.82
0.95
Positively co-operative
3.64
0.93
Positively co-operative
DGEECDCGEE 1.01 x 1011 M-2
1.85
0.89
Positively co-operative
1.92 x 1020 M-4
3.82
0.97
Positively co-operative
pEEEEGDD
76
2.5
1/qM (mmol/g)-1
2.0
apoLMWCr
EDGEECDCGD
DGEECDCGDE
pEEEEGDD
1.5
1.0
0.5
0.0
0
500
1000
1500
2000
1/[M] (mM-1)
Figure 3.13. Langmuir isotherm for Cr(III) uptake by apoLMWCr and synthetic peptides
at 4 °C.
77
Table 3.3. Langmuir isotherm parameters of apoLMWCr and synthetic peptides
Samples
Langmuir isotherm
Bt (mmol/g)
K (L/mmol)
R2
Apo-LMWCr
-2.69
-109.44
0.95
EDGEECDCGE
-6.97
-353.59
0.97
DGEECDCGEE
-62.89
-9.94
0.99
pEEEEGDD
-0.73
-151.09
0.96
78
6
5
1/qM (mmol/g)-1
4
3
2
1
0
0
10
20
30
40
50
60
70
(1/[M]qM)1/2 (g/mM*mmol)1/2
Figure 3.14. Langmuir isotherm (BM0.5 model) of Cr(III) adsorbed on apoLMWCr,
Volume of adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial
pH: 7.4.
79
50
1/qM (mmol/g)
-1
40
30
20
10
0
0
100
200
300
400
500
600
700
(1/[M]qM)1/2 (g/mM*mmol)1/2
Figure 3.15. Langmuir isotherm (BM0.5 model) of Cr(III) adsorbed on EDGEECDCGE,
Volume of adsorption medium: 200 ml , temperature: 4 °C, adsorption time: 12 h, initial
pH:7.4.
80
[a]
0.020
Absorbance
0.015
0.010
0.005
0.000
300
400
500
600
700
800
wavelength (nm)
[b]
0.8
Abs
0.6
0.4
0.2
0.0
240
260
280
300
320
340
360
380
400
wavelength (nm)
Figure 3.16. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-EDGEECDCGE
complex. [Cr] = 0.03 mM.
81
[a]
0.020
0.015
0.010
0.005
0.000
-0.005
-0.010
300
400
500
600
700
800
wavelength (nm)
[b]
1.2
1.0
Abs
0.8
0.6
0.4
0.2
0.0
240
260
280
300
320
340
360
380
400
wavelength (nm)
Figure 3.17. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-DGEECDCGEE
complex. [Cr] = 0.04 mM.
82
[a]
0.020
Abs
0.015
0.010
0.005
0.000
300
400
500
600
700
800
wavelength
[b]
0.10
0.08
Abs
0.06
0.04
0.02
0.00
240
260
280
300
320
340
360
380
400
wavelength
Figure 3.18. Visible spectrum (a) and ultraviolet spectrum (b) of Cr-oligopeptide
pEEEEGDD.
83
3.6
Conclusions
Chromium-binding studies indicate that the synthetic peptides EDGEECDCGE and
DGEECDCGEE bind 2 equivalents of chromic ions while pEEEEGDD binds 4 equivalents of
chromic ions per peptide. The binding of chromium is highly cooperative for all the peptides.
The Hill binding constants at physiological pH for EDGEECDCGE and pEEEEGDD are on the
order of 1023 and 1020, respectively, and are close to that of apoLMWCr. The binding of Cr to
pEEEEGDD may be quite similar to that of Cr to apoLMWCr given the Cr bound to both appear
to be in similar pseudo-octahedral ligand fields. All binding constants above suggest that the
sequence of pEEEEGDD seems a better candidate for apoLMWCr than either EDGEECDCGE
or DGEECDCGEE.
84
REFERENCES
(1)
Clodfelder, B. J.; Vincent, J. B. Journal of Biological Inorganic Chemistry 2005, 10,
383-393.
(2)
Sun, Y. J.; Ramirez, J.; Woski, S. A.; Vincent, J. B. Journal of Biological Inorganic
Chemistry 2000, 5, 129-136.
(3)
Vincent, J. B. Journal of the American College of Nutrition 1999, 18, 6-12.
(4)
Vincent, J. B. Journal of Trace Elements in Experimental Medicine 2003, 16, 227-236.
(5)
Jacquamet, L.; Sun, Y. J.; Hatfield, J.; Gu, W. W.; Cramer, S. P.; Crowder, M. W.;
Lorigan, G. A.; Vincent, J. B.; Latour, J. M. Journal of the American Chemical Society
2003, 125, 774-780.
(6)
Dinakarpandian, D.; Morrissette, V.; Chaudhary, S.; Amini, K.; Bennett, B.; Van Horn, J.
D. BMC Chemical Biology 2004, 4, Article No.: 2.
(7)
Chesneau, V.; Becherer, J. D.; Zheng, Y. F.; Erdjument-Bromage, H.; Tempst, P.; Blobel,
C. P. Journal of Biological Chemistry 2003, 278, 22331-22340.
(8)
Wei, P.; Zhao, Y. G.; Zhuang, L.; Ruben, S.; Sang, Q. X. A. Biochemical and
Biophysical Research Communications 2001, 280, 744-755.
(9)
Fritsche, J.; Moser, M.; Faust, S.; Peuker, A.; Buttner, R.; Andreesen, R.; Kreutz, M.
Blood 2000, 96, 732-739.
(10)
Davis, C. M.; Vincent, J. B. Archives of Biochemistry and Biophysics 1997, 339, 335-343.
(11)
Lin, T. W.; Huang, S. D. Analytical Chemistry 2001, 73, 4319-4325.
85
(12)
Gode, F.; Pehlivan, E. Bioresource Technology 2007, 98, 904-911.
(13)
Gao, J. Ph.D. dissertation, University of Alabama, 2008.
(14)
Gode, F.; Pehlivan, E. Journal of Hazardous Materials 2006, 136, 330-337.
(15)
Masel, R. I. Principles of Adsorption and Reaction on Solid Surfaces; Wiley Interscience:
New York, 1996.
(16)
Cornish-Bowden, A. Principles of Enzyme Kinetics; Butterworths: London, 1976.
(17)
Shiner, J. S.; Solaro, R. J. Biophysical Journal 1984, 46, 541-543.
(18)
Aksoyoglu, S. Journal of Radioanalytical and Nuclear Chemistry-Articles 1990, 140,
301-313.
(19)
Subramaniam, V.; Hoggard, P. E. Journal of Coordination Chemistry 1994, 31, 157-165.
(20)
Schiewer, S.; Wong, M. H. Environmental Science & Technology 1999, 33, 3821-3828.
(21)
Levina, A.; Codd, R.; Dillon, C. T.; Lay, P. A. Progress in Inorganic Chemistry, Vol 51
2003, 51, 145-250.
86
4.
CHAPTER 4
SEQUENCE LMWCr BY MS
4.1
Introduction
Elucidating a potential role for chromium in enhancing insulin sensitivity has been
hindered by experimental problems. The LMWCrs isolated from animal liver and human urine
are assumed to be identical or nearly so based solely on their similar molecular weight, amino
acid composition and chromium-binding potential (refer to Chapters 2 and 3). To date, LMWCr
has not been proven to be antigenic, preventing its presence to be detected using immunological
techniques. The absence of sequence and structural information limits progress in the field. Mass
spectrometry (MS) is currently the best available experimental method that might yield high
resolution sequence information on LMWCr, after the failure of conventional Edman
degradation techniques.1 From the MS fragmentation pattern of a peptide, its amino acid
sequence can be deduced. In principle, fragmentation of a peptide may yield six different
sequence-specific ion series (a, b, and c type from N-terminus, and x, y, and z type from Cterminus determined) (Scheme 4.1),2 but not all ions are formed with equal facility. If the
LMWCr sequence can be confirmed, a peptide with this sequence could be synthesized and used
to potentially produce an antibody that might cross-react with biological LMWCr.
The success attributed to identification and characterization of peptides separated after
liquid chromatography (LC) by MS is highly dependent on the purity of the end-product. One of
87
88
Scheme44.1.
Scheme44
1 Peptide fragmentation notation using the scheme of Roepstorff and Fohlman
Fohlman. (provided by Dr.
Dr
Cassady)
the factors limiting the detection of peptides is the presence of nonvolatile low molecular weight
contaminants (e.g., salts), frequently observed when analyzing peptides purified by
chromatography; these result in adduct formation, increased chemical noise and pronounced ion
suppression effects. Ion desalting and concentration of a peptide with introduction of a reversedphase (RP) column prior to mass analysis usually result in increase a signal-to-noise ratio and
sensitivity.3
Unfortunately, the properties of LMWCr make it difficult to characterize. LMWCr
appears to be a naturally occurring Cr-binding oligopeptide composed of glycine, glutamate,
aspartate and cysteine with the carboxylates comprising more than half of the total amino acid
residues. This composition has a marked negative contribution to retention in RP columns which
reduce retention of LMWCr in column. No potential signal from LMWCr or its fragments
signals were identified by MS after applying and attempting to elute from ZipTipTM (Millipore)
or Isolute HAX SPE columns, performed according to the manufacture’s protocol. Such a small,
extremely hydrophilic acidic peptide with an estimated isoelectric point (IEP) of about 3.17 is
affected by ion suppression more strongly than other molecules when it occurs in
desorption/ionization in MS, which results in this type of peptide isolated from biological source
rarely being detected in MS.4-6 Even when MS or tandem MS spectra of LMWCr have been
acquired, unambiguous confirmation of every m/z feature to a corresponding fragment is
difficult because Cr is retained by many peptide fragment ions and bound to multiple sites of the
peptide to “hold it together”, and fragments with Cr bound lose varying quantities of protons.7
Generating apoLMWCr by treating with EDTA and a reducing agent followed by separation
with Sephadex G-15 chromatography has been successful, but trace amounts of EDTA remain.
We have found that the EDTA dominates the mass spectra of the product. Recently graphite
89
powder (GP) has been utilized to effectively retain small and hydrophilic peptides, which could
be readily eluted for MS analysis.8
This chapter will describe attempts to sequence and confirm the amino acid composition
of LMWCr by MS.
4.2
Instrumentation
MALDI-MS were performed on a Bruker Daltonics (Billerica, MA, USA) Reflex III
MALDI/TOF mass spectrometer with delayed extraction. Spectra were obtained in positive or
negative reflector mode and positive or negative linear mode using an accelerating voltage of 20
kV. Post-source decay (PSD)9 fragment ion spectra were processed under negative voltage after
isolation of the appropriate precursor ion by using timed ion selection. The peptide precursor ion
was isolated by timed ion selection. Fragment ions were detected in segments by stepping down
the reflectron voltage as following: -21.0, -19.55, -15.75, -11.82, -8.86, -6.64, -4.98, -3.74, and 2.80 KV. Laser focus, laser power, and detector gain are ramped up for later segments to
optimize signal. The individual segment spectra were then stitched together in the processing
software (Flexanalysis) to give a PSD spectrum.
For on-line LC-MS analysis of LMWCr, the Agilent 1200 series liquid chromatography
with Zorbax (150 × 0.5 mm) 5B-C18 column was interfaced to a Bruker HCT ultra PTM
discovery system ion trap mass spectrometer (Billerica, MA, USA) via an ESI probe. The mobile
phase A was dH2O, and mobile phase B was acetonitrile. A gradient elution at a flow rate of 10
µl/min was performed as follows: 0-5 min 2% B (isocratic), 5-44 min 2% - 80% B (linear
gradient), and then 44-60 min 100% B (isocratic). The spray needle voltage was 4 kV; the ion
transfer capillary temperature was 300 °C. The mass spectra were obtained within a range of m/z
100-2000 in negative mode. Tandem mass spectrometry employed low energy collision-induced
90
dissociation (CID).10 The CID collision gas is helium. The fragmentation amplitude was 1.0 V,
with smart fragmentation on (the start Amplitude 30% and the end amplitude 200%). The datadependent tandem mass spectrometry (MS/MS) experiments were controlled using the menudriven software provided with the Bruker system.
4.3
Materials
α-Cyano-4-hydroxycinnamic acid (4HCCA), trifluoroacetic acid (TFA) and activated
charcoal (C-5510) were obtained from Sigma. LC/MS grade acetonitrile was obtained from
Riedel-de Haën. The LMWCr’s of alligator, bovine, chicken live and human urine were purified
with methods discussed in chapter 2. The peptides pEEEEGDD and pEEEGEDD were
synthesized in the Cassady group of The University of Alabama.
4.4
Methods
4.4.1
Graphite Powder Microcolumn
Custom-made chromatographic microcolumns were used for desalting and concentration
of the peptide prior to MS analysis. Activated charcoal was packed in a constricted GELloader
tip (Eppendorf). A 10-mL syringe was used to force liquid through the column by applying
gentle air pressure. The columns were equilibrated with 10 µL of 0.1% TFA. An aliquot of the
LMWCr after purification by Sephadex G-15 column chromatography was diluted to 30 µL in
0.1% TFA and loaded onto the column using gentle syringe air pressure. The column was
washed with 60 µL 0.1% TFA, and peptides were eluted directly onto the MALDI target with
approximately 2 µL of 4HCCA in 70% acetonitrile/0.1% TFA.
In order to generate more samples, ESI-MS samples were prepared with a modified
method in which activated charcoal powder was packed into a microconcentrator instead of the
GELoading tip. A tabletop centrifuge was used to wash the sample and elute the sample through
91
charcoal powder and filter membrane. 70% acetonitrile/0.1%TFA was used for the final elution
solution. Elutent was lypholyzed and re-dissolved in dH2O before LCMS processing. Amino acid
analyses of samples were performed by the Protein and Separation Analysis at Laboratory at
Purdue University.
4.4.2
Immunization
GenScript Corporation performed the immunization procedures using the sequences
CEEEEGDD and CEEEGEDD, which were deduced from MSn spectra. The immunization
process involved covalently bonding the antigen to keyhole limpet hemocyanin (KLH), through
a disulfide bond, and periodically injecting the conjugated protein into an animal, in this case,
rabbits. The peptides have an extra cysteine residue at the N-terminus to use in forming the
disulfide linkage. After four weeks from the initial injection, blood is collected from the rabbits,
and the serum is tested for antibodies. A second and final bleed at week six and eight from the
initial injection, respectively, was carried out. Serum from these bleeds was also tested. Enzymelinked immunosorbent assay (ELISA) tests were followed with peptide-BSA conjugate as
ELISA antigen.
4.5
Results and Discussion
4.5.1
MALDI-TOF MS Studies
Only one molecular peak (m/z 804) was observed for all treated LMWCr samples with
GP column under negative mode after subtracting background noise (Figure 4.1).
No
corresponding m/z peak was found under positive mode. The Y-axis shows the absolute value of
the intensity of the ion signals. The low signal intensities possibly resulted from poor binding
capacity of the microcolumn or inefficient elution using 70% acetonitrile/0.1% TFA, or the
samples not being ionized well. Amino acid analysis of bovine LMWCr elutes from GP columns,
92
G:E:D:C::1.0:4.5:2.2:0, indicated that the LMWCr has lost some of its amino acids during GP
microcolumn processing. Determination of the Cr:peptide ratio was inaccessible possibly
because application of 0.1% TFA impeded the fluorescamine assay for peptide concentration by
blocking the N teminus. However, the Cr content in the eluate decreased so dramatically that Cr
could not be detected by the diphenylcarbazide method.
PSD of the m/z 804 ion of bovine LMWCr was performed (Figure 4.2) and two
sequences are proposed, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate). All m/z
peaks match to predicted ions by allowing m/z ±1 difference except the feature at m/z 804 is +2
larger than expected. The sequence pEEEEGDD was utilized to measure its chromic ion binding
constants of chromic ion in chapter 2. PSD spectrum of synthetic peptides were generated and
compared to these of the LMWCr’s (Figure 4.3). The spectra of the LMWCr’s from different
sources shared several common features at m/z 384, 428, 482, 570 and 759, which suggest a
similarity on sequence. However, the PSD of peptide pEEEEGDD showed only a few similar
features at m/z 428, 482, 570 to those of the LMWCr’s, while that of pEEEGEDD was quite
different from LMWCr’s in MALDI-TOF PSD MS. More identifiable fragments and information
on low m/z area were required to further studying the possible sequence of LMWCr by MALDITOF PSD.
4.5.2
Analysis of LMWCr using LC-MS with ESI
Larger samples of LMWCr were isolated by the modified GP column with the
application of the microconcentrator. The ability of RP column (Zorbax 5B-C18 on LC/MS) to
retain LMWCr was tested; experiments revealed that the fragments of LMWCr may elute during
the first 5 mins when washing column with 2% acetonitrile; these were detected by obvious UV
absorbances at 260 nm (Figure 4.4). No ESI response (m/z 802) could be observed because
93
ionization interferences occur when an extract from a biological specimen, LMWCr in this case,
is loaded into the LC portion of the instrument. Suppression of the signal at the time point that
corresponds to the void volume of the column is common.11 Off-line ESI-MS was adapted for
detection of bovine LMWCr ions with an infusion injection. One peak at m/z 802 and another at
m/z 401 corresponding to the singly and doubly charged species, respectively, were observed
under only negative mode. These exactly conform to the MW of pEEEEGDD or pEEEGEDD
(Figure 4.5). Low-energy CID MS/MS (MS2) at m/z 802 ions and MS/MS/MS (MS3) at m/z 784
ions were carried out on both ions to identify their sequence, while the synthetic peptides
pEEEEGDD and pEEEGEDD were fragmented under the same conditions (Figures 4.6-4.8).
Fragments under MS/MS and MS/MS/MS provide more potential sequence information. Ions
belonging to pEEEEGDD and pEEEGEDD were found in the CID spectra of LMWCr at m/z
802 and 401. Very similar spectrum features were shared by LMWCr and the two synthetic
peptides. A little difference was observed in MS3 spectra of m/z 784 ions between the two
synthetic peptides. Peaks with m/z 392 and 397 are found only in the spectra of pEEEEGDD,
while a feature m/z 632 is only in that of pEEEGEDD (circle marked in Figure 4.7). The features
at m/z 392 and m/z 397, but not at m/z 632, are found in the spectrum of the fragment of bovine
LMWCr, suggesting that the sequence of the fragment may be pEEEEGDD. However, a final
conclusion about the sequence of the LMWCr sequence is difficult to make without a throughout
explanation of all the m/z peaks.
Spectra from MALDI-TOF MS and ESI-MS both indicate that no Cr exists in the
fragment. The combination of GP and TFA may contribute to this result in which bound Cr was
stripped under low pH solution with 0.1% TFA through ion-exchange then activated charcoal
94
could chelate the cofactor (Cr) from LMWCr to make the fragment apoLMWCr.12,13 The
reaction
Cr-O2CR + HO2CR’ ----HO2CR + Cr-O2CR’ eqn. (1)
occurs if HO2CR’ is more acidic than HO2CR, where the symbols R and R' denote an attached a
hydrocarbon side chains. The retention mechanisms of GP include electron donor-acceptor
interactions in addition to hydrophobic interaction. The retention of anionic compounds (such as
the apoLMWCr fragment in this case) is dominated by electronic interactions between the solute
and the delocalized electron clouds on the graphitized carbon, while other compounds may be
positive charged under low pH (~3) and mainly retained by reversed-phase interaction with the
hydrophobic carbon surface. Anionic and cationic compounds may be separated with a mobile
phase containing an organic modifier (such as acetonitrile) and an electronic modifier (such as
TFA) for elution.14
4.5.3
Immunization and bioinformatics
Both Peptide-KLH conjugation products produced significant quantities of antibody,
suggesting these synthetic peptides have reasonable antigenicity (Table 2). Cross interaction
between LMWCr’s and antibodies will be tested in the near future to determine the selectivity of
the antibodies and whether they can be used to develop an assay for LMWCr (see Table 2).
In vivo, some peptides with bioactivity originate from the processing of proteins, such as
laminin and hemorphins gamma gain, that are fragments of functional protein domains and play
roles in the regulation of cellular pathways.15,16 A genomic search against NCBI non-redundant
(nr) base with sequence EEEEGDD was performed to find proteins containing this sequence
motif. Multiple 100% hits were found due to the short sequence and low complexity: seven
sequences in Homo sapiens; two in Bos taurus; two in Gallus gallus; one in Mus musculus. None
95
of these was reported to be a responsive gene in chromium treatment of human lung epithelial
(A549) cells and mouse testis (TM4 Sertoli-like) cells.17,18 The lack of a match may be a result of
LMWCr being produced at greater levels in hepatic cells than in other tissues.19
96
Human
urine
804.129
600
400
200
0
804.605
500
400
300
200
100
0
Bovine
liver
832.103
804.689
804.603
1200
1000
800
600
400
200
0
650
Alligator
liver
814.825
800
600
400
200
0
700
750
800
Chicken
liver
850
900
950
m/z
Figure 4.1. Signals with m/z ~804 peak appear in the spectra of all treated LMWCr’s with GP
column in negative mode MALDI-TOF MS.
97
1500
.
[EEGD-H]- or [EGED-H]-?
[M-H]-
1250
1000
618
750
500
c3
-
[b4483
-NH3]-*
a5-
[M-H-CO2]-
-
c5
c6-
250
0
200
300
400
500
600
700
800
m/z
Figure 4.2. MALDI-TOF PSD of m/z 804 ion of Bovine LMWCr in 70% ACN/0.1%
TFA/10% 4HCCA.
98
(a)
804.213
(b)
803.907
(c)
(d)
(e)
800.962
(f)
801.953
Figure 4.3. MALDI-TOF PSD of m/z 804 ions of (a) alligator, (b) bovine, (c) chicken, (d)
human urine and synthetic peptides, (e) pEEEEGDD and (f) pEEEGEDD.
99
Intens.
x107
uc1107__Cr_g25_2nd_gp_negative_1-F,5_01_253.d: TIC -All MS
8
6
4
2
0
Intens.
[mAU]
uc1107__Cr_g25_2nd_gp_negative_1-F,5_01_253.d: UV Chromatogram, 260 nm
200
150
100
50
0
0
10
20
-MS, 3.6-3.9min #(242-260)
Intens.
5
x10
30
40
50
60
70
632.2
2.5
80
Time [min]
-MS, 3.6-3.9 min
376.1
2.0
190.8
673.2
1.5
395.9
110.8
1.0
753.1
0.5
848.1
0.0
200
400
600
800
1000
1200
1400
m/z
Figure 4.4. Elution profile on Zorbax 5B-C18 column in dH2O and acetonitrile
(linear gradient). (top) Total Ion Concentration (TIC), (middle) UV chromatogram
at 260 nm, and (bottom) spectra of MS between 3.6 to 3.9 minutes.
100
101
200
208.2
250.7
260.7
pEEEGEDD
309.4
300
289.7
pEEEEGDD
220.8
270.7
Bovine LMWCr
343.1
336.1
327.0
381.1
368.9
400
400.7
400.6
400.5
424.0
445.7
444.7
544.2
510.4 525.7 543.0
500
490.7
463.0 479.1
562.2
600
594.2
634.2
634.0
655.2
671.2
687.3
700
699.8
687.2
684.9
673.2
671.2
745.3
733.1
763.3
759.1
800
802.3
784.3
802.3
781.7
802.3
m/z
835.3
Figure 4.5. ESI-MS (top) of bovine LMWCr treated with GP processing, (middle) of pEEEEGDD and (bottom) of
pEEEGEDD.
0
2
4
6
8
0
x10 6
1
2
3
0
x10 7
2
4
6
) Intens.
x10 4
102
0
0
200
400
600
800
1000
1000
2000
3000
4000
5000
Intens.
100
c1
-
[pD-H]-
200
[EG-H2O] ?
-
″b2-
2-
2-
300
2
2-
2-
2-
400
2-
500
3
c5-
[c5-H2O] -
″a5-?
600
700
[c6-H2O] -
m/z
Figure 4.6. Low CID spectra of (top) MS of m/z ~401 and (bottom) MS of m/z ~391 from bovine LMWCr samples.
Double charged peaks are marked with "2-".
103
200
238.9
238.9
pEEEEGDD
223.9
pEEEGEDD
223.7
238.9
300
295.0
294.9
Bovine LMWCr
313.0
313.2
314.0
353.2 367.4
348.1
368.0
400
397.0
392.1
410.1
410.1
410.0
425.1
428.2
429.1
443.1
464.1
482.1
482.1
482.1
500
526.1
554.2
553.1
544.2
542.9
535.2
526.1
514.2
508.2
526.2
571.2
572.3
583.1
572.1
600
603.1
604.5
625.2
632.2
651.3
651.2
651.1
669.2
669.3
669.2
687.2
700
696.3
687.2
698.2
687.2
722.2
722.2
720.2
740.3
740.2
766.2
766.2
766.2
757.2
Figure 4.7. Low CID ESI-MS/MS (top) of bovine LMWCr treated with GP processing, (middle) of pEEEEGDD and
(bottom) of pEEEGEDD
0
1
2
3
4
5
0.005
x10
0.25
0.50
0.75
1.00
1.25
1.50
0
x10 6
2000
4000
6000
Intens.
783.3
m/z
785.2
785.0
104
100
pEEEGEDD
pEEEEGDD
[pD-H]-
200
Bovine LMWCr
223.9
223.9
223.9
b2 -
263.9
300
295.1
295.0
295.0
313.1
313.1
312.9
353.2
353.0
410.1
400
392.2
410.1
397.1
392.0
410.1
464.1
482.1
464.2 482.1
464.1 482.2
500
508.2
526.2
526.2
553.2
544.1
̈a5-?
[y5-H2O]-
571.3
572.2
c5-
600
618.2
632.2
[c6-H2O]-
̈b5[c6-2H2O]650.2
650.2
669.0
669.2
700
[M-D-H]
686.3
722.3
705.1 722.3
704.0
b6 -
748.2
748.4
738.2
-
Figure44.8. Low CID ESI-MS/MS/MS (top) of bovine LMWCr treated with GP processing, (middle) of pEEEEGDD and
(bottom) of pEEEGEDD
0
2000
4000
6000
0
1000
2000
3000
0
1000
2000
3000
Intens.
[M-2H2O-H]
m/z
766.2
766.3
Table 4.1. ELISA results of rabbit’s serum immunization with synthetic peptides as
immunogens.
Dilution/Absorbance Pre-immunization serum
(A450 nm)
Antiserum
immunization
(A450nm)
CEEEEGDD
CEEEGEDD
CEEEEGDD
CEEEGEDD
1:1000
0.082
0.076
2.007
1.806
1:4000
0.076
0.070
1.052
1.097
1:16000
0.074
0.069
0.552
0.602
1:64000
0.067
0.061
0.160
0.267
1:256000
0.061
0.055
0.076
0.114
blank
0.055
0.068
0.052
0.059
•
Data of CEEEEGDD come from the 4th immunization while data of CEEEGEDD
come from the 5th immunization.
105
Three of the seven hits in the Homo sapiens genome are parts of apoptotic chromatin
condensation inducer 1 (ACIN1) protein. Reported peptides isolated from the chromatin mostly
show common biochemical features which also are possessed by LMWCr fragment:
low
molecular weight from 600 to 1000 Da; pyroglutamic acid blocking the N-terminus; predominant
presence of glutamic acid and aspartic acid. Most of them show high affinity for the protein
kinase CKII, which can provoke phosphorylation of insulin receptor (IR) and also phosphorylate
IR substrate IRS-1, which reinforces the possible connection of this protein kinase with the
functional elements involved in transmission of the insulin signal.19-21
4.6
Conclusion and Future Work
Microcolumns packed with graphite powder (GP) (activated charcoal powder) were
tested to examine the potential for this relatively inexpensive material to be used as an alternative
to RP material to desalt and concentrate LMWCr prior to MS. A GP column treated with 0.1%
TFA were shown to strip Cr from LMWCr and make an apoLMWCr fragment. MS showed the
fragment to be comprised of 1pE, 3E, 1G, and 2D. Comparison of MS spectra with peptides of
the two possible sequences of the fragment, pEEEEGDD and pEEEGEDD, revealed that the
former is the most likely sequence of the fragment, which also is consistence with the conclusion
of Chapter 3. Antibodies have been obtained to peptides of the sequences EEEEGDD and
EEEGEDD. The sensitivity and specificity of the antibodies will be tested to determine if they
can be used to probe for LMWCr in tissue samples and body fluids.
Additional MS experiments, especially CID MSn on m/z 410, will hopefully confirm the
sequence of the fragment and help us to explore the ion type deduced by the acidic oligopeptide
in this catalog.
106
NMR studies of the fragment may also aid in confirming the sequence and establishing
the binding site of the fragment. 2D NMR technology with double quantum filtered correlation
spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), hetero-nuclear multiple
quantum coherence (HMQC), hetero-nuclear multiple bond coherence (HMBC) and nuclear
Overhauser effect spectroscopy (NOESY) can be applied to solve the sequence of the peptide if a
0.3 mM peptide fragment sample can be obtained. A chemical shift perturbation study can be
carried out by NMR to identify the binding site of Cr ions on the peptide. Cr ions at different
concentrations may bind to the peptide specifically, and different 2D 15N-heteronuclear single
quantum coherence (HSQC) spectra can be expected based on the result that there are possibly 4
Cr ions bound to the peptide fragment. By comparing the chemical shift changes before and after
the addition of Cr ion, the affected amino acid residues will be monitored, and thus the metal
binding site can be established.
107
REFERENCES
(1)
Davis, C. M.; Vincent, J. B. Archives of Biochemistry and Biophysics 1997, 339, 335-343.
(2)
Roepstorff, P.; Fohlman, J. Biomedical Mass Spectrometry 1984, 11, 601.
(3)
Zhang X.; Picariello W.; Hosein N.; Towle M.; Goetzinger W. Journal of
Chromatography A. 2006, 30, 147-155.
(4)
Black, S. D.; Mould, D. R. Analytical Biochemistry 1991, 193, 72-82.
(5)
Hirabayashi, A.; Ishimaru, M.; Manri, N.; Yokosuka, T.; Hanzawa, H. Rapid
Communications in Mass Spectrometry 2007, 21, 2860-2866.
(6)
Meek, J. L. Proceedings of the National Academy of Sciences of the United States of
America 1980, 77, 1632-1636.
(7)
Pu, D.; Vincent, J. B.; Cassady, C. J. Journal of Mass Spectrometry 2008, 43, 773-781.
(8)
Larsen, M. R.; Cordwell, S. J.; Roepstorff, P. Proteomics 2002, 2, 1277-1287.
(9)
Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Communications in Mass
Spectrometry 1992, 6, 105-108.
(10)
Cody,R. B.; Freiser, B. S. Analytical Chemistry 1982, 54, 1431-1433.
(11)
Bonfiglio, R.; King, R. C.; Olah, T. V.; Merkle, K. Rapid Communications in Mass
Spectrometry 1999, 13, 1175-1185.
(12)
Jin, X. S.; Foley, K. M.; Geiger, J. H. Journal of Biological Chemistry 2004, 279, 1388913895.
(13)
Wildgoose, G. G.; Leventis, H. C.; Davies, I. J.; Crossley, A.; Lawrence, N. S.; Jiang, L.;
Jones, T. G. J.; Compton, R. G. Journal of Materials Chemistry 2005, 15, 2375-2382.
108
(14)
Gu, G.; Lim, C. K. Journal of Chromatography 1990, 515, 183-192.
(15)
Hibino, S.; Shibuya, M.; Engbring, J. A.; Mochizuki, M.; Nomizu, M.; Kleinman, H. K.
Cancer Research 2004, 64, 4810-4816.
(16)
Karelin, A. A.; Blishchenko, E. Y.; Ivanov, V. T. Neurochemical Research 1999, 24,
1117-1124.
(17)
Ye, J. P.; Shi, X. L. Molecular and Cellular Biochemistry 2001, 222, 189-197.
(18)
Cheng, R. Y. S.; Alvord, W. G.; Powell, D.; Kasprzak, K. S.; Anderson, L. M.
Reproductive Toxicology 2002, 16, 223-236.
(19)
Calzuola, I.; Giavarini, F.; Sassi, P.; De Angelis, L.; Gianfranceschi, G. L.; Marsili, V.
Peptides 2005, 26, 2074-2085.
(20)
Angiolillo, A.; Bramucci, M.; Marsili, V.; Panara, F.; Miano, A.; Amici, D.;
Gianfranceschi, G. L. Molecular and Cellular Biochemistry 1993, 125, 65-72.
(21)
Trujillo, R.; Miro, F.; Plana, M.; Jose, M.; Bollen, M.; Stalmans, W.; Itarte, E. Archives
of Biochemistry and Biophysics 1997, 344, 18-28.
109