•
Essentials
in
HPLC '88
Poster Presentation
Isolation of Thymosin B4 from Thymosin
Fraction 5 by Reverse Phase HPLC
M. Badamchian,
Waters.bioresearchThe
for
absolute,
essential
M.P. Strickler, M.J. Stone, A.L. Goldstein
WotersChromotography
Milford34Millip°re
givisiOnMapleMAS,
Corporotion
01757
(508)478-2000
War
Divisiooof M,LL,PoREeFs ,/
ISOLATION
OF THYMOSIN
MAHNAZ
BADAMCHIAN
ALLAN
L. GOLDSTEIN
B
4
FROM
+, M. PATRICIA
+
THYMOSIN
FRACTION
5 BY RP-HPLC
STRICKLER W. M. JUDE STONE W. AND
+ Department of Biochemistry,
The George Washington University
School of Medicine and Health Sciences. Washington. DC 20037
w Waters Chromatography
Division. Millipore Corporation.
Life
Science Application Laboratory, Fairfax. VA 22030
ABSTRACT
THYMOSIN B 4 (TB4) IS ONE OF THE BIOLOGICALLY
ACTIVE PEPTIDES
IN THYMOSIN FRACTION 5 (TF5). A PARTIALLY PURIFIED THYMIC PREPARATION
FROM CALF THYMUS.
TB 4 HAS HORMONE-LIKE
IMMUNE AND NEUROENDOCRINE
RESPONSES.
WE HAVE DEVELOPED
A RAPID
TWO-STEP
PROPERTIES
METHOD
AND CAN MODULATE
FOR THE PURIFICATION
OF TB 4 FROM TF5.
THIS PURIFICATION
IS BASED ON THE USE OF HIGH
PERFORMANCE SEMI-PREPARATIVE
AND ANALYTICAL REVERSED-PHASE
(CIB DELTA PAK) CHROMATOGRAPHIC
COLUMNS.
AMINO
ANALYSIS AND POLYACRYLAMIDE
GEL ELECTROPHORESIS
ACID COMPOSITIONAL
HAVE SHOWN
THAT THE PURIFIED
TB 4"
TB 4 IS IDENTICAL
TO SYNTHETIC
t
I,
INTRODUCTION
A PARTIALLY PURIFIED THYMOSIN PREPARATION FROM CALF THYMUS TERMEO
THYMOSIN FRACTION 5 (TF5} HAS BEEN STUDIED EXTENSIVELY FOR BIOLOGICAL
ACTIVITY. AS WELL AS IN VIVO AND IN VITRO IMMUNE RESPONSES.
TF5 CONSISTS
OF A FAMILY OF BIOLOGICALLY
ACTIVE POLYPEPTIDE COMPONENTS WITH HORMONE-LIKE
ACTIVITIES.
THYMOSIN B4 (TB4) IS ONE OF THE SEVERAL PEPTIDES THAT IS
PRESENT IN TF5 AND PARTICIPATES
IN THE PROCESS OF REGULATION. DIFFERENTIATION
ANO FUNCTION OF THYMUS-OEPENOENT
THYMOCYTES. TB4 IS COMPOSED OF 43 AMINO
ACID RESIDUES AND HAS A MOLECULAR WEIGHT OF 4982 AND A PI OF 5.i. THE
AMINO-TERMINUS
OF THE PEPTIDE IS BLOCKEO BY AN ACETYL GROUP.
TB4 HAS BEEN
PURIFIED FROM TF5 BY CONVENTIONAL
COLUMN TECHNIQUES FOR LARGE-SCALE
PURIFICATION
USING ION-EXCHANGE
CHROMATOGRAPHY
ON A CARBOXYLMETHYLCELLULOSE COLUMN FOLLOWED BY GEL FILTRATION ON SEPHADEX G-50 IN 6 M
GUANIDINIUM-HCI.
THE DESALTED TB4 ON SEPHADEX G-iO HAD A YIELD OF 0.45Z.
TB4 HAS ALSO BEEN ISOLATED BY HPLC TECHNIQUES WITH A VERY LOW RECOVERY,
2-3%, USING A uBONDAPAK CiB COLUMN.
LOW RECOVERIES HAVE ALWAYS BEEN A
PROBLEM IN THE ISOLATION OF TF5 PEPTIDES AND HAVE LIMITED THE AMOUNT OF
PURIFIED PEPTIDE AVAILABLE FOR FURTHER CHARACTERIZATION
AND COMPARISON
OF THE DIFFERENT FORMS OF THE BIOLOGICALLY ACTIVE PEPTIDES.
IN THE
PRESENT STUDY WE REPORT A VERY FAST. REPRODUCIBLE
AND EFFICENT LARGE SCALE
PROCEDURE AS WELL AS AN ANALYTICAL RP-HPLC PROCEDURE FOR THE PURIFICATION
DF TB4 FROM TF5.
RP-HPLC
OF THYMOSIN
ANALYTICAL
FRACTION
5
SEPARATION
1400 -
J
0
J
o!!,,,,V
5
10
15
20
25
30
35
40
45
50
55
,
60
MINUTES
FIGURE I. REVERSED PHASE HPLC SEPARATION OF go0 UG OF THYMOSIN FRACTION 5
(TF5) ON A 3.9 MM X 30 CM DELTA PAK. 300 A. 15 u. C18 COLUMN.
BUFFER A
WAS 0.02 M AMMONIUM ACETATE. pH 6.8. AND BUFFER B ACETONITRILE.
A 60
MINUTE LINEAR GRAOIENT FROM 0-80% B WAS RUN AT A FLOW RATE OF 0.5 ML/MIN.
DETECTION WAS AT 280 NM, 1.4 AUFS.
;
RP-HPLC
OF THYNOSIN
PREPARATIVE
FRACTION
5
SEPARAT ION
i50O -
J
0
>
t
I
I
5
iO
i5
|
EO
O
25
O
I
I
O
|
I
!
30
35
40
45
50
55
60
MINUTES
FIGURE 2.
REVERSED PHASE HPLC SEPARATION OF i.5 G OF THYMOSIN FRACTION 5
(TF5) ON A 50 MM X 30 CM DELTA PAK. 300 A. 15 u, CiB COLUMN.
BUFFER A
WAS 0.02 M AMMONIUM ACETATE. pH 6.8. AND BUFFER B ACETONITRILE.
A 60
MINUTE LINEAR GRADIENT FROM 0-80% B WAS RUN AT A FLOW RATE OF BO ML/MIN.
DETECTION WAS AT 2BO NM AT 1.5 AUFS. COLLECTED FRACTIONS WERE ASSAYED
FOR THYMOSIN B4 USING SOLID-PHASE RIA.
RESULTS ARE OVERLAID ON THE
CHROMATOGRAM.
ANALYTICAL
RP-HPIC
I
OF FRACTION
I
20
I
I
tO0
mv
BO
BO
40 -
|
0
!
w
|
i
•
J
i
i
|
!
iO
!
!
m
|
•
w
|
e
|
e
•
2O
¢
v
i
u
u
w
u
|
•
3O
w
|
e
e
,
|
|
i
|
e
i
|
i
4O
Minutes
FIGURE 3.
REVERSED PHASE HPLC SEPARATION OF 130 UG OF FRACTION 20
FROM THE 1.5 G SEPARATION OF THYMOSIN FRACTION 5 ON A 3.9 MM X 15 CM
DELTA PAK. 300 A. 5 u. CIB COLUMN.
BUFFER A WAS 0.1% PHOSPHORIC ACID
AND BUFFER B ACETONITRILE WITH 0.1% PHOSPHORIC ACID.
SEPARATION OF
THYMDSIN B4 WAS ACHIEVED USING A iO MINUTE LINEAR GRADIENT FROM 0-14% B .
FOLLOWED BY A 10 MINUTE HOLD AT 14% B.
A I0 MINUTE LINEAR GRADIENT TO
18% B ELUTED THE THYMOSIN B4 (1). THE FLOW RATE WAS 1 ML/MIN.
DETECTION
WAS AT 214 NM. O.i AUFS,
D
PREPARATIVE
RP-HPLC
I
OF FRACTION
I
20
I
I
lO00
mV
BOO
600
400
2O0
0
0
iO
20
30
40
Minutes
FIGURE
4.
FROM THE
REVERSED
i.5
PHASE HPLC
G SEPARATION
300 A, 5u,
C18 COLUMN.
ACETONITRILE
WITH O.iZ
USING A
HOLD AT
WAS i
tO MINUTE
14% B AND
ML/MIN.
WERE ASSAYED
OVERLAID
OF TF5
OF 5.6
ON A 3.9
WAS AT
2i4
B4 USING
CHRONATOGRAM.
NM,
SOLID
CM DELTA
PHOSPHORIC
SEPARATION
LINEAR
GRADIENT
FROM 0-i4%
A iO MINUTE
LINEAR
GRADIENT
DETECTION
MG OF FRACTION
MM X i5
BUFFER A WAS 0.i%
PHOSPHORIC ACID.
FOR THYMOSIN
ON THE
SEPARATION
i
AUFS.
PAK,
ACID AND BUFFER
WAS ACHIEVED
B, FOLLOWED
TO 18% B.
B
BY A iO MINUTE
THE FLOW RATE
COLLECTED
PHASE RIA.
20
FRACTIONS
RESULTS
ARE
h
RECHROMATOGRAPHY
OF FRACTION
I
I
33
I
I
m
500
mV
400
300
I
m
200
iO0
L
.
. _
_A__
o
0
iO
20
30
40
Minutes
FIGURE 5. RECHROMATOGRAPHY
OF iO0 UL OF FRACTION 33 FROM THE PREPARATIVE
SEPARATION OF FRACTION 20.
THE SAMPLE WAS DILUTED WITH iO0 UL OF WATER
AND INJECTED ON TO THE 3.9 MM X 15 CM DELTA PAK, 300 A, 5 u, CiB COLUMN.
BUFFER A WAS 0.1% PHOSPHORIC ACID AND BUFFER B ACETONITRILE
WITH 0.1%
PHOSPHORIC ACID.
A iO MINUTE LINEAR GRADIENT FROM 0-14% B, FOLLOWED BY
A iO MINUTE HOLD AT 14% B AND A iO MINUTE LINEAR GRADIENT TO IB% B
ELUTED THE THYMOSIN B4 (1). THE FLOW RATE WAS I ML/MIN.
DETECTION WAS
AT 214 NM, 0.5 AUFS.
"
RECHROMATOGRAPHY
OF FRACTIONS
I
I
33
& 33
I
5
I
25O
mV
D
20O
1
150
100
5O
0
0
10
20
30
40
Minutes
FIGURE
OF
AND
6.
FRACTION
RECHROMATOGRAPHY
33.
INJECTED
BUFFER
THE
A iO MINUTE
SAMPLE
ON TO THE 3.9
A WAS 0.1%
PHOSPHORIC
OF
WAS
A 10 MINUTE
HOLD
AT
ELUTED
THE
THYMOSIN
AT 214
NM,
0.5
AUFS.
14_ B AND
B4
(I).
33
ACID
TO
CM DELTA
PAK,
AND BUFFER
LINEAR
FLOW
RATE
FROM
250
300
UL.
A,
THE
FROM 0-t4%
LINEAR
WAS
GRADIENT
I ML/MIN.
SEPARATION
DILUTED
5 u,
B ACETONITRILE
GRADIENT
A iO MINUTE
THE
_ 33.5
CONCENTRATED
MM X t5
PHOSPHORIC
ACID.
FRACTIONS
C1B
WITH
B,
TO
WITH
WATER
COLUMN.
0.1%
FOLLOWED BY
i8_
B
DETECTION
WAS
CRUDE
SYNTHETIC
B4
VS
ISOLATED
B4
1
20O
mV
150
m
t00
0
I
I
I
I
I
I
I
l
I
I
I
I
200
mY
150
100
0
I
0
5
_0
I
t5
Minutes
I
20
I
25
30
35
40
FIGURE 7.
COMPARISON OF 50 UG OF CRUDE SYNTHETIC B4 AND B4 ISOLATED
BY THE TWO STEP RP-HPLC PROCEDURE ON THE 3.9 MM X 30 CM DELTA PAK.
300 A. 5u COLUMN USING THE 6RAOIENT DESCRIBED PREVIOUSLY
(FIGURE 6).
THE SYNTHETIC B4 WAS IDENTIFIED BY SOLID PHASE RIA AND THE RESULTS ARE
OVERLAID ON THE CHROMATOGRAM.
RED IS SYNTHETIC B4. BLUE IS ISOLATED B 4
AND BLACK IS CPM FROM THE RIA.
J
IW
I
FIGURE 8. SDS-PAGE OF SYNTHETIC AND ISOLATED THYMOSIN B4
ABOUT 10-20 UG OF THYMOSIN B4 SAMPLES WERE ELECTROPHORESEO
IN A 1.5 MM 12% SDS-POLYACRYLAMIOE
GEL ACCORDING TO THE
METHOD OF LAEMMLI
(1970) AND STAINED USING A BIOIRAO
SILVER STAIN KIT.
LANE I: MOLECULAR WEIGHT MARKERS FROM
BIO-RAD; LANES 2 AND 3 SYNTHETIC AND NATURAL THYMOSIN B4
RESPECTIVELY.
#
•
AMINO ACID COMPOSITION a OF NATURAL AND SYNTHETIC THYMOSIN B4
b
AMINO ACID
SYNTHETIC B4
NATURAL B4
REPORTED SEQUENCE
Asp
Glu
Ser
GIy
Thr
Ala
Pro
Met
Ile
Leu
Phe
Lys
a.
3.9
i0.8
3.5
i.i
2.8
2.2
2.9
0 .B
i .6
i.6
0.9
B.5
3.8
i0.6
3 .B
i.2
3.2
i .B
2.9
0.7
i .9
2.2
O.B
B.B
4
ii
4
i
3
2
3
i
2
2
i
9
The data are presented as assumed numbers of residues
per molecule,
b.
Number of residues obtained from the
reported sequence
{Low and Goldstein, i9Bi, Proc. Natl.
Acad. Sci., 78, ii62).
TABLE i. AMINO ACID ANALYSIS - THIS WAS PERFORMED USING
THE PICO TAG AMINO ACID ANALYSIS SYSTEM OF WATERS CHROMATOGRAPHY
DIVISION, MILLIPORE. ABOUT iO UG OF SYNTHETIC AND ISOLATED TB4
SAMPLES WERE HYDROLYZED WITH 6 N HCf CONTAINING I% PHENOL BY
VOLUMEAT iiO°C FOR 48 HRS. THE HYDROLYSATESWEREDRIED AND
USED FOR AMINO ACID ANALYSIS BY THE PICO TAG STANDARDPROCEDURE
[Bidlingmeyer,
et al.
(t984)
J. Chromato_r.
336,
94-104].
l
CONCLUSION
I.
WE WERE ABLE TO PURIFY TB4 FROM TF5 USING A TWO-STEP RP-HPLC
TECHNIQUE WITH AN 80% RECOVERY FOR EACH STEP.
THIS PURIFICATION
WAS BASED ON THE RIA AND HPLC RETENTION TIME OF THE SYNTHETIC B4.
THE FINAL TB4 PREPARATION
IS HOMOGENEOUS AND THERE IS A 30-FOLD
INCREASE IN ITS RECOVERY COMPARED TO PREVIOUSLY REPORTED
PURIFICATION PROCEDURES FOR TB4 FROM TF5 (i-2}.
2.
SDS-PAGE
REVEALED
3.
THE NATURAL
COMPOSITION
OF THE ISOLATED AND SYNTHETIC TB4, AFTER SILVER STAINING,
A SINGLE PEPTIDE BAND WITH A MOLECULAR WEIGHT BELOW iO KD.
TB4 HAD A SIMILAR HPLC
AS THE SYNTHETIC B4.
GENERAL
L.
Low,
T.
2.
Low,
T. L. K., and Mercer,
3.
Low. T. L. K., Hu, S. K., and Goldstein,
78,
and
Goldstein,
I162.
TIME
AND AMINO
ACID
REFERENCES
1.
Acad. Sci.,
K.,
RETENTION
A.
R. C.
L.
(i962)
(1984)
J.
Biol.
Chem.
J. Chromatogr.
A. L.
257.
301,
(IgBi} Proc.
t000.
221.
Natl.