117
.
C O L U M N CHROHATOGRAPHY OF P R O T E I N S ,
LIPOPROTEINS AND L I P I D S
MILTON
E, B A I L E Y
Historical Introduction.
Although there were many e a r l y contributors t o the development of
column chromatography, two pioneers a r e considered the f a t h e r s of t h i s
analytical technic. They a r e David Talbot Day (1859-1925) and Mikhail Tswett
(1872-1919). Day was a graduate chemist who became a distinguished geologist
and mining engineer. He suggested the idea of chromatography t o the F i r s t
International Petroleum Congress and the Geological Society of Washington
i n 1900 and 1903 respectively. Day passed o i l through f u l l e r s earth and
fractionated it into saturated aliphatic hydrocarbons, aromatic and unsaturated substances and f i n a l l y , nitrogen and sulfur compounds.
T s w e t t , a Russian, studied botany and physical chemistry a t the
University of Geneva, then returned t o Russia. He demonstrated the p o t e n t i a l
use of chromatography by separating xanthophylls and chlorophyllins using
pet ether as a solvent and calcium carbonate as adsorbant. Tswett’s achievement was superior t o Day’s i n two respects. F i r s t , he recognized and
correctly interpreted chromatographic processes, and second, he devised a
useful laboratory method. The o r i g i n a l Tswett column is s t i l l widely used
for both preparative and analytical purposes.
Basic Nomenclature.
Tswett recognized t h e importance of chromatography and devised the
nomenclature which i s most widely used today (see figure 1) The tube
f i l l e d with the adsorbent was called the chromatographic column. The washi n g l i q u i d was called the solvent, wash l i q u i d o r developer. The s e r i e s of
zones in t h e column was the chromatogram and t h e washing of t h e mixture t o
form the chromatogram was referred t o as development.
Contemporary terminology has replaced adsorbent with sorbent,
sorptogram f o r chromatogram and sorptographic analysis f o r chromatographic
analysis (1), and has introduced terms such as elution t o mean a type of
development when the solutes a r e washed through t h e columns and collected.
Eluate indicates t h e solvent fraction plus solute collected in t h e f i l t r a t e .
It i s customary t o distinguish between three main types of chromatographic
procedures8 elution analysis, f r o n t a l analysis, and displacement analysis.
Since we w i l l be primarily concerned with elution analysis i n t h i s discussion, t h e other two types of chromatography w i l l not be mentioned further.
Column Chromatography of Proteins.
The column chromatography of proteins has been t r e a t e d i n nmerous
review a r t i c l e s and books (2-15). The i n s t a b i l i t y of proteins i n the
presence of t h e usual type of adsorbent has l e d t o the development of three
118
I
substantially different types of adsorbents f o r proteins. These are calcium
phosphate gels, cellulose and Sephadex. The f i r s t two have general applicab i l i t y t o column chromatography of proteins (2).
Calciwn phosphate gels have f o r some time been widely used i n the
purification of enzymes by batch procedures, but, i n c o l w work, t h e
extremely high resistance imposed by such material t o t h e flow of aqueous
buffers has been a serious handicap. However, higher flow r a t e s have been
made possible by mixing the g e l with Super-Cel, o r by forming the g e l i n
t h e presence of cellulose (16 and 1 7 ) . Recently, methods have been
developed for t h e preparation of caloium phosphate i n forms which permit
adequate flow without t h e addition of f i l t e r aids. One of these consists
chiefly of brushite (CaHP04. 2H20) (18). Another i s hydroxylapatite
ca,(P04)30H (19)
The cellulose adsorbents are available as e i t h e r anion o r cation
exchangers, and both types have been used i n the chromatography of proteins
(9 and 20-23). The most commonly employed cellulose ion-exchangers a r e
diethylaminoethyl cellulose, an anion exchanger and carboxymethyl cellulose,
a cation exchanger. Many others have also been used (Table I).
Recently ion-exchange Sephadex, the cross linked dextran, has been
used i n t h e separation o f proteins and polypeptides. Undoubtedly both ionexchange and g e l f i l t r a t i o n phenomena are involved i r ? t h i s type of
separation (24-27).
Elution of Proteins from Ion-Exchangers.
Although basic studies of the adsorption of proteins on ion-exchange surfaces are lacking, t h e formation of multiple e l e c t r o s t a t i c bonds
between t h e protein and the adsorbent i s undoubtedly involved. Both t h e
protein and the ion-exchanger are polyelectrolytes, and they are, therefore,
capable of interacting a t several points, provided interchange distances are
favorable. Breover, experience shows that the adsorbed protein i s more
l i g h t l y bound than a singly charged substance under t h e same conditions, but
can be eluted from a suitable ion-exchanger if the pH i s changed t o reduce
t h e number of charges on the protein or adsorbent, o r i f t h e salt concentrat i o n i s raised t o compete f o r the existing charges.
The eluting buffer i s usually selected f o r i t s effectiveness i n
controlling pH i n t h e region in which chrornqbgraphy i s t o be conducted.
Two types of elution are used f o r t h e separation of proteins on
ion-exchange columns.
1. Stepwise elution
2.
Gradient elution
In the former, a r b i t r a r y stepwise changes i n t h e composition of the eluent
a t short volume i n t e r v a l s can be used t o e l u t e the protein rapidly and i n
r e l a t i v e l y high concentration. Eowever, resolution can generally be expected
t o be less than that attainable by gradient elution.
\
119
8
An example of stepwise elution of protein applied t o t h e meats
f i e l d i s some recent work of Fujimaki and Deatherage (28), who are i n t e r ested i n t h e chromatographic fractionation of sarcoplasmic proteins of beef
skeletal muscle on ion-exchange cellulose. One hundred gram samples of
f r e s h l y slaughtered beef were extracted with d i s t i l l e d water and 900 mg of
the extract chromatographed on cellulose phosphate. Fourteen fractions were
separated on t h e column ( 2 x 40 cm); they were dialyzed, centrifuged and
freeze dried. The samples were f i n a l l y analyzed f o r enzymic a c t i v i t y . The
r e s u l t s a r e shown i n Table 11. Fraction I was considered t o be nucleoprotein. This was determined by the identification of nucleotides following
hydrolysis and chromatography on Dowex-1, X-8 (formate form).
Fraction IV and VI were found t o be oxymyoglobin and metmyoglobin
from t h e i r absorption spectra. Aldolase a c t i v i t y was associated with
f r a c t i o n s I11 through IX. Phosphopyruvic kinase, a c t i v i t i e s localized i n
f r a c t i o n s VI, VI1 and IX and A’llPase and Cathepsin a c t i v i t i e s found in Fraction
I X . Since e s s e n t i a l l y a l l a c t i v i t i e s were found i n F’raction VI, protein i n
t h i s fraction was rechromatographed on DEAE-cellulose.
Similar studies involving t h e fractionation of protein i n rabbit
muscle were reported at t h i s conference last year (29).
Another example of stepwise elution involved a very simple method
similar t o t h a t of Mitz and YanaxI (30). T h i s procedure w a s used t o study
pork cathepsins (31). The apparatus used f o r t h i e study i s shown i n f i g u r e 2.
Twenty-f ive grams of DEAE-cellulose were used i n a column (2.5 x 25 cm) The
exchanger was washed with carbon dioxide-free water and 100 mg of a sample
previously fractionated with armnonium sulfate (50-60$ cut). The i n i t i a l
solvent was C 0 2 f r e e deionized water followed by C02-saturated deionized water.
Figure 3 shows t h e type of separation obtained with protein from pork.
.
A similar method has been used by Sliwinski and co-workers (32) i n
studies of beef cathepsins.
Numerous studies of proteins u t i l i z i n g column chromatography have
involved gradient elution. A commonly employed hydro-dynamic apparatus i s
pictured i n figure 4, taken from Yaguchi e t a l . (33). Gradient elution involves e i t h e r a change i n ionic strength o r pH of t h e eluting solution.
Chamber I contains s t a r t i n g buffer and i s connected t o one o r more chambers
i n series containing solutions of d i f f e r e n t pH o r ionic strength.
--
Figure 5 i s a gradient elution chromatographic apparatus used at
Missouri; it includes a gradient system similar t o t h a t shown f n the previous
figure and a rather large chromatographic column (12 x 4.5 cm) of DEAE-cellulose. The effluent i s passed through a c e l l and the absorbancy i s automatic a l l y recorded by a Vanguard (figure 6 ) .
An example o f t h i s type of chromatography involves the separation
of cathepsins B and C by Landmum (34) who used CM-cellulose and a gradient
of phosphate buffer as eluent. Sephadex G-25 was used t o separate protein
from a beef extract which was further purified by adsorption on DEAE-cellulose
i n t h e absence of carbon dioxide. The a c t i v e proteins were extracted by
lowering t h e pH s l i g h t l y w i t h carbon dioxidesaturated water. The extracted
protein was then chromatographed on a CM-cellulose column using a phosphate
120.
gradient from 0 t o 0.05 M. Results are shown i n f i g u r e 7. The f i r s t peak
contained cathepsin C and a small amount of cathepsin B. The second peak
had an a c t i v i t y which resembled cathepsin B, but t h i s peak did not appear i n
a muscle preparation t r e a t e d according t o t h e usual procedures f o r the
p u r i f i c a t i o n of cathepsin B. Peak 3 is cathepsin B and corresponds t o t h e
peak of highest a c t i v i t y i n a regular cathepsin B preparation.
hblecular Sieve F i l t r a t i o n ( Sephadexl
The three-dimensional network of a g e l has a decisive influence on
the diffusion of dissolved substances. This f a c t i s u t i l i z e d t o obtain
separations where t h e s i z e of the s o l u t e i s an importasit parameter. I n a
column packed with small swollen g e l p a r t i c l e s , solutes of a large molecular
s i z e a r e excluded from t h e g e l and emerge from t h e c o l u m without retardat i o n while solutes capable of diffusing i n t o t h e i n t e r i o r of t h e p a r t i c l e s
a r e retarded. The method i s c a l l e d g e l f i l t r a t i o n o r molecular sieve filtration. Such a g e l can be made with Sephadex, small grains of hydrophilic in*
soluble substance made by cross-linking t h e polysaccharide dextran. The network has a non-ionic character and t h e polar properties a r e almost e n t i r e l y
due t o t h e high content of hydroxyl groups,
This process i s excellent f o r separation of low molecular w e i g h t
materials from p r o t e i n s o r for t h e separation of proteins of d i f f e r i n g
molecular weights. It has had many uses in the study of proteins (35-42) and
some use i n the m e a t s f i e l d . Ann DuFresne (personal communication), AMIF', is
using Sephadex G-75 t o separate hemoglobin from myoglobin. The myoglobins
are f u r t h e r separated in CM-cellulo se i n t o three components.
Column Chromatography of Lipoproteins.
Column chromatography i s not a very popular a n a l y t i c a l procedure
f o r separating lipoproteins because so many c l a s s i c a l methods a r e available.
These include : d i f f e r e n t i a l s a t p r e c i p i t a t i o n (43), mving boundary
electrophoresis (44), ultracentrifugation (45), zone electrophoresis (46),
alcohol fractionation (47), complex formation with dextrin (48), as w e l l as
the other a n a l y t i c a l technics t h a t w i l l be described by speakers t o follow.
One adsorbent used i s powdered glass. This material has been used
as adsorbent i n a column f o r t h e separation of lipoproteins of blood
serum. A t y p i c a l chromatogram i s shown i n f i g u r e 8. There are two f r a c t i o n s
of t h e alphal-lipoprotein type, i.e., with a cholesterol, phospholipid r a t i o
below 1. The f i r s t one i s not adsorbed a t pH 8.8 and the second I s eluted a t
pH 9.4. Above pH 9.4, ltpoproteins of t h e beta-type with a cholesterol,
phospholipid r a t i o above 1, are eluted.
(49)
Column Chromatography of Lipids.
The last 10 y e a r s have seen great s t r i d e s i n l i p i d research,
l a r g e l y as a r e s u l t of t h e development o r adaptation of various forms of
chromatography. Many reviews of t h e application of chromatography t o t h e
l i p i d f i e l d have recently appeared (see reference 3, page 451), and the
subject i s w e l l treated in two text books on t h e subject (50-51).
A maJor portion of t h e developments i n t h e column chromatography
of l i p i d s involves adsorption technics, and t h e elution technics described
121.
f o r proteins also apply t o l i p i d s . Adsorbents as cellulose (52), charcoal
(53 and 54), alumina (55) and f l o r i s i l (56) have been used successfully i n
the past, but s i l i c i c acid has become t h e mst effective and popular
adsorbent f o r t h e separation of l i p i d s .
A n extensive review of t h e chromatography of l i p i d s on s i l i c i c
acid and a detailed discussion of the preparation and properties of these
adsorbents have been given by Wren (57). By far the most important applicat i o n of s i l i c i c acid adsorption chromatography has been the separation of
classes of l i p i d s and, i n particular, t o the further subfractionation of
phospholipids
Since Trappe's introduction t o s i l i c i c a c i d chromatography f o r
l i p i d separations i n 1940 ( 5 8 ) , considerable experience has been gained by
t h i s procedure. Because of the great p o t e n t i a l use of s i l i c i c acid chromatography and because of i t s apparent u n r e l i a b i l i t y on occasions, Hirsch and
Ahrens (59) made a systematic study of t h e many variables and suggested a
standardized method of preparing the adsorbent, and packing and conditioning the columns and eluting l i p i d mixtures. These investigators used both
stepwise and gradient elution technics.
Figures 9 and 10 are pictures of t h e chromatographic equipment
used at the Missouri s t a t i o n f o r l i p i d fractionations. Hirsch and Ahrens
found t h a t t h e most successful elution of l i p i d s i n t o classes was carried
out w i t h a solvent mixture of increasing polarity. The solvent p a i r used
was e t h y l ether and pet ether (b.p. 60-70) Samples were applied i n amounts
up t o 300 mg (but containing l e s s than 50 mg of any one component) t o a
column of 18 g of s i l i c i c acid. Reproduclble separations of n e u t r a l l i p i d
classes from one another and from phospholipids were achieved with fixed
amounts of mixtures of petroleum ether and diethyl ether by increasing the
proportion of diethyl ether stepwise and by f i n a l elution with methanol.
The method proved reliable i n the chromatography of various synthetic and
natural l i p i d mixtures.
An elution diagram of the l i p i d components of human plasma i s
shown i n figure ll. Lipid content was determined gravimetrically, cholest e r o l esters and cholesterol was identified by the Libermann-Burchard reaction, non-esterified f a t t y acids were determined by reaction t o bromcresol
green. Phospholipids were determined by phosphorous analysis. The last
three peaks contained cephalin, l e c i t h i n and sphingomyelin respectively.
After separation, other methods may be used f o r further separation
and study of t h e l i p i d classes. S i l i c i c acid has been used i n more thorough
study of the lipoproteins and phospholipids (60) and paper chromatography
seems t o be p a r t i c u l a r l y adaptable t o the analysis of phospholipid (61).
G a s and t h i n layer chromatography are being used t o study many classes of'
l i p i d s t h a t might f i r s t be i s o l a t e d using s i l i c i c acid column chromatography.
I n f r a red spectroscopy i s a l s o used t o identify and study chromatographic
f r a c t i o n s (62).
Hornstein and co-workers (63) used s i l i c i c acid columns t o
separate phospholipid from neutral fat. The columns were developed with
successive 300 m l portions of chloroform-methanol 20:1, chloroform-methanol
1:l and methanol. They were also concerned with the nature and concentration
122.
of free f a t t y a c i d s present i n cured and cooked meats and with t h e i r possible
e f f e c t s on f l a v o r (64). They found t h a t n a t u r a l l y occurring free f a t t y acids
i n meat could be determined i n the presence of l a r g e amounts of unsaponified
f a t by adsorbing t h e free f a t t y a c i d s on a strong anion base exchange resin
(amberlite IRA-400) and washing the r e s i n f r e e of fat with p e t ether and
converting the free f a t t y acids t o t h e i r methyl e s t e r s d i r e c t l y on the r e s i n
w i t h anhydrous methanol-HC1.
The nature and concentration of the f a t t y
acids were then determined by gas chromatography.
Recovery data was presented f o r f r e e f a t t y acid mixtures added t o
10 m l . of 576 f a t emulsions and was based on the recovery of n-heptadecanoic
a c i d as an i n t e r n a l standard. The average recovery f o r 7, CZl8 f a t t y acids
(lauric, myristic, palmitic, s t e a r i c , o l e i c , l i n o l e i c , and l i n o l e n i c ) was
102.2$. Absolute recoveries ranged f r o m 60-95$.
McCarthy and Duthie (65) found that t h i s method could be used
quantitatively provided t h e Amberlite r e s i n IRA -403 was used and recharged
four o r f i v e times. They found, however, t h a t best results could be obt a i n e d by using a column procedure employing s i l i c i c acid t r e a t e d with
isopropanol-KOH. After adsorption of t h e l i p i d m i x t u r e on t h e column, t h e
n e u t r a l l i p i d s were eluted with e t h y l ether. The f a t t y acids could then be
removed from the column with 50 ml. of 2$ formic a c i d i n e t h y l ether followed
by 75-100 m l of e t h y l ether. Phospho-lipids were retained on the column.
The results of 26 analyses of f a t t y acids mixed with various other l i g i d a
showed an average recovery of 98.3$ f o r free f a t t y acids. The range was
from 95.5 t o l03.8$.
Table I11 shows the d i s t r i b u t i o a and recovery of C14 labeled
l i p i d s after separation on KDH-treated s i l i c i c acid. These investigators
a l s o used i n f r a r e d spectrophotometry t o show t h a t t h e glyceride f r a c t i o n
w a s f r e e of carboxyl absorption while the free f a t t y acid f r a c t i o n s were
f r e e of e s t e r s .
It i s extremely informative and nearly obligatory t o examine all
column f r a c t i o n s during t h e separation of l i p i d s by some other a n a l y t i c a l
method i n order t o determine their purity. The speakers t o follow i;i;tloutl i n e some of these procedures f o r you.
In conclusion, I would l i k e t o say t h a t t h e use of column
chromatography i s in f t s infancy i n regard t o i t s use a6 a t o o l i n separating t i s s u e components of meat. I hope t h a t t h i s paper and i t s bibliography
w i l l a c t as a crutch t o speed t h e use of t h i s important a n a l y t i c a l technic
t o maturity i n studies of t h e many ramifications of our ever growing field.
123.
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I. Chemical composition of four fractions. J. Lab. and Clin.
Med., 48, 36 (1956).
Fractionation of polypeptides and p r o t e i n s on dextran
Clin. Chim, Acta., 4, 776 (1959).
-
-
scow,
1961.
-
-
46
Kunkel, N. G. and R. J. Slater. Lipoprotein p a t t e r n s of serum
obtained by zone electrophoresis. J. Clin. Invest., 31, 677
c
(1952)
47.
Gurd, F. R. N., S. L, Oncley, J. T. Edsall, and E. J. Cohn. The
lipoproteins of human plasma. Disc. Farady %c., 5, 70 (1947).
48
Cornwell, D. G. and F. A. Kruger. Molecular complexes i n t h e
i s o l a t i o n and characterization of plasma lipo-proteins.
J. Lipid Research, 2, 110 (1961).
-
49.
Carlson, L. A. Chromatographic separation of serum lipoproteins on
glass powder colwnns. i n I I I r d I n t e r . Conf. Biochem. Problems
Of Lipids, pp. 113-122 71956)-
50
Kaufmann, H. D.
(1958)
51.
Hanahun, D. J.
Analyse der F e t t e usd Fettprodukte, Berlin, Springer
Lipide Chemistry.
New York, Wiley
(1960).
52 e
Smith, R. H. The phosphatides of t h e latex of Hevea b r a s i l i e n s i s .
Biochem. J., 57, 130 (1954),
53.
Claesson, S. Frontal analysis and displacement development i n
chromatography. Annals. N. Y. Acad. Sei. 49, 183 (1948)
-
,
.
54. Holman, R. T. and W. T. W i l l i a m s . Displacement analysis of l i p i d s .
J. Am. Chem. Soc., 73, 3337 and 5285 (1951).
-
55.
Wrgan, E. D. and N. Polgar. Constituents of l i p i d s of Tubercle
Bacilli. Part V I I I . Studies on myculic acid. J. Chem. SIC.,
3779 (1957).
See a l s o ibid. 1902 (1958).
56
Carroll, K. K. Separation of l i p i d classes by chromatography.
J. Lipid Res., 2(2), 135 (1961).
57.
58
.
59.
-
-
Wen, J. J.
-4,
Chromatography of l i p i d s on s i l i c i c acid.
1 7 3 (1960).
Trappe, W.
-
Biochem. Z., 305, 150
J e
Chromatog.,
--
(l940), ibid. 306, 316.
Hirsch, J. and E. H. Ahrens, Jr. The separation of complex l i p i d
mixtures by t h e use of s i l i c i c a c i d chromatography. J. Biol.
Ch@m., 233, 311 (1958).
-
60. Nelson, G. J. Studies on human serim lipoprotein, phospholipids and
phospholipid f a t t y acid composition by s i l i c i c acid chromtography. J. Lipid Res., 3(1), 71 (1962)
-
61
62
63
64
Martnetti, G. V. Chromatographic separation, i d e n t i f i c a t i o n and
analysis of phosphatides. J. Lipid Res.,
1 (1962).
Anderson, D. M. W. Application of infra red spectroscopy. The
i&?a&ification and determination of chromatographic f r a c t i o n s
Analyst, a4, 50 (1959).
Hornstein, I., P. F. Crowe and M. J. Heimberg. Fatty acid
composition of neat t i s s u e l i p i d s . Je Food Sci., 26, 581
(1961)
Hornstein, I., J. A. Alford, L. E. E l l i o t t and P. F. Crowe.
Determination of f r e e fatty acids i n fat. Anal. Chem., 32
540 (1960).
7 ,
65
McCarthy, R. D. and A. H. Duthie. A rapid quantitative method for
t h e separation of free f a t t y acids from other l i p i d s . J. Lipid
Res., 2 ( 1 ) , 117 (1962).
127.
f i g u r e 1. The chromatographic column. The tube filled with
adsorbent i s c a l l e d t h e chromatographic column. The washing
l i q u i d i s c a l l e d t h e solvent o r developer. The series o f
zones ( s o l u t e s ) i s c a l l e d t h e chromatogram.
128.
Table I
Cellulose Ion Exchangers
Anion Exchangers
DEAE-Cellulo se
(strongly+basic t e r t i a r y amine,
-O-C,$4-N
H (C2H5) $
1
' )
T W - C e l l u l o se
( s t r o n g l y b a s i c quaternaly amine,
-0- ( CzH4-N+ ( C2H5) gC1')
AE-Cellulose
( s l i g h t l y b a s i c amine,
-o-czI14.NH2)
GE-Cellulo se
( s t r o n g l y basic,
-O-C2H~.~.C=~2.r~2+Cl-)
ECTEOLA-Cellulo se
(mfxed amines, C1")
Cation Exchangers
- -
CM- Cellulo se
(high capacity f o r b a s i c and n e u t r a l
p r o t e i n s , -0 C H ~COOH)
P-Cellulo se
( s t r o n g l y a c i d i c , -0-P03H2)
SE-Cellulose
(strongly acidic,
-O-C~H~SO~'€F
PAB- Ce l l u l o se
( d i a z o t i z a t i o n + coupling,
-0-CH -C H -NH2)
2 6 4
h e m i c a l and enzymic properties of freeze-dried,
ongissimus dorsi
fractionated sarcoplasmic protein from beef
from Fujimaki and Deatherage (28)
7
pH of elution buffer
Table XI
Ppt. Solub.
with in
T U 1 water
Enzymic
O.D.(3mg/5 ml
activity
28ommS 26Omms Aldolase LDH PPK m k .
-
ATPase
-
-
+
0.200
0.340
+
0.321
0.265
-
+
+
0.268
0.202
++
++
+
0;398
0.388
+
- -
++
-
-
-
-
7
+
+
++
7
+
4-
++
4-+
Cathep.
Rem
Nucleopr
-
-
-
-(mu)
++
630 (me--
5
glob
+
c,
++
++
++
++
+
+
+
7
+
+
00212
0.169
++
8
c
+,
0.370
9
+
0.321
t
0,306
0.236
9
%CA = Trichloracetic acid
-
-
t,
+-?
130.
f i g u r e 2. Equipment used i n study of pork cathepsins.
Twenty-five grams, DEAF,-Cellulose i n a column (2.5 x 25 cm).
I
I
i
2 4 6 810 14 1810
30
40
-
__
50
J',
1
60
70
80
lRMll0W WUMBll
IW
90
110
I10
f i g u r e 3. Elution diagram of ammonium s u l f a t e (50-60$)
soluble pork sarcoplasmic proteins. Elution with C02-free
water and COZ-saturated wzter. Largest peak contained
g r e a t e s t mount of c athepsi n a c t i v i t y
From P a r r i s h (31).
.
131.
Chamber
m
Chombcr II Chomber
I
f i g u r e 4. Hydrodynamic apparatus
f o r gradient e l u t i o n column
chromatography. Chamber I contains
s t a r t i n g b u f f e r and i s coriected t o
one o r more chambers i n s e r i e s cont a i n i n g s o l u t i o n s of d i f f e r e n t p H
o r i o n i c strength.
From Yaguchi e t al. (33).
100-110 em.
--
2 3-25cm.
Elution BufferDEAL-
I
I
To Fraction Collector
f i g u r e 5. Gradient e l u t i o n chromatographic apparatus used
t o separate proteins. D m - C e l l u l o s e c o l w n ( 4 . 5 x 12 cm).
132.
f i g u r e 6.
CMC
Vanguard automatic u l t r a v i o l e t analyzer.
ELUTION DIAGRAM OF PROTEASE F R A C T I O N D - 2
O
0.0
''I
OD.
1
0.41
h
-I\. .-----
I P/ml .
wo
100
400
LOO
f i g u r e 7. CM-Cellulose separation of beef cathepsins previously p u r i f i e d
by DEAE-Cellulose adsorption and e l u t i o n with COZ-saturated water. Phosphate
g r a d i e n t between 0 and 0.05 M. Peak a t 100 ml contained cathepsin C and
small amount of cathepsin B. Peak a t 300 ml had a c t i v i t y resembling
cathepsin B. Peak a t 1100 ml had cathepsin C a c t i v i t y .
From Landmann (34).
133.
0
5
io
15
20
25
Fraction number
30
35
40
f i g u r e 8. Chromatographic separation of serum l i p o p r o t e i n s on powdered
glass. F i r s t two peaks c o n t a i n a l p h a r l i p o p r o t e i n (cholesterol/phosphol i p i d (1). Above pH 9.4 b e t a - l i p o p r o t e i n s (cholesterol/phospholipid 1)
a r e eluted.
From Carlson ( 4 9 ) .
>
figure 9.
E l u t i o n apparatus and s i l i c i c a c i d column f o r l i p i d analysis.
134.
f i g u r e 10.
Column chromatographic apparatus f o r l i p i d analysis.
Flarnm hprde -lU0 rng
a Lsbesrnann-Burchard
reactson
95
m la
f i g u r e 11. Stepwise e l u t i o n diagram of blood plaslca l i p i d s on 18 gm
s i l i c i c a c i d cclumn. The a b s c i s s a shows t h e tube number c o l l e c t e d
f r a c t i o n a l l y and t h e various solvents used t o e f f e c t e l u t i o n .
From Hirsch and Ahrens (59).
135.
Table I11
D i s t r i b u t i o n and Recovery of C14-Labeled Lipids
A f t e r Separation on H3H-Treated S i l i c i c Acid
McCarthy and Duthie (65)
Labeled Substance
Added*
Control
Activity '
CPm
A c t i v i t y Recovered
cpm
Fraction
Fraction
1
2
Tripalmitin- 1-C14
171,400
178,900
0
Cholesterol-4-C14
369,280
360,720
900
Chol e s te r yl
pa~aitate-l-~l'
657,360
667,920
660
Palmitic a ~ i d - l - 6 ~
185,600
0
185,200
Linoleic acid- 1-$4
100,490
0
96,270
Butyric a ~ i d - 1 - C ~ ~
233,250
0
220,000
-
*Mixed with glycerides and FF'A p r i o r t o separation
-'An a l i q u o t
equivalent t o t h a t separated
MR. PEARSON:
Thank you, D r . Bailey, f o r a very informative
talk.
Following r i g h t along i n t h e l i n e of Colunm Chromatography t h e r e
i s another technique t h a t i s being extensively used i n research, p a r t i c u l a r l y i n t h e f i e l d of l i p i d s and t o some extent on other d e r i v a t i v e s a f i'ecting p r o t e i n s , t h a t i s Gas Chromatography. T h i s morning we have Dr.
H. B. Craig of North Carolina S t a t e College who i s going t o d i s c u s s t h i s
t o p i c f o r us.
#if##########