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Purification and sequence study of platelet aggregation inhibitor from snake venom

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Purification and sequence study of platelet aggregation inhibitor from snake venom

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Content Purification and Sequence Study of Platelet Aggregation
Inhibitor from Snake Venom
by
Chung-Kang Ho
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 1994
Copyright 1994 Chung-Kang Ho
UMI Number: EP41848
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
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a note will indicate the deletion.
Dissertation Publishing
UMI EP41848
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
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789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6
U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA
THE GRADUATE SCHOOL
U N IV E R S ITY PARK
LOS ANGELES, C A LIFO R N IA 9 0 0 0 7
This thesis, w ritten by
Chung-Kang Ho
under the direction of h.±s. Thesis Com m ittee,
and approved by all its members, has been p re
sented to and accepted by the Dean of The
Graduate School, in p a rtial fu lfillm e n t of the
requirements f o r the degree of
Dm, June 16, 1994
THESIS COMMITTEE
Dean
Chairman
ACKNOWLEDGEMENTS
I would like to thank Dr. Francis S. Markland for
instruction and guidance. I would also like to thank Mohit
Trikha and Simy Loayza for technical assistance, and Ms.
Ginny Kortes, R.N., USC Comprehensive Center, for providing
phlebotomy services required for these studies. Finally, I
would like to appreciate my parents and families for
supporting and encouraging during these past years.
CONTENTS
Page
List of Figures iv
Abstract V
Introduction 1
Experimental Procedure 4
Results 9
Discussion 23
References 26
List of Figures
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Page
1. Purification of multisquamatin by hydrophobic
interaction chromatography (HIC). 10
2. Purification of mutisquamatin by reverse-phase
chromatography (I)............................... 11
3. Purification of multisquamatin by reverse-phase
chromatography (II).............................. 12
4. Purification of multisquamatin by CM300 cation-
exchange chromatography.......................... 13
5a. Multisquamatin on C18 analytic column....... 14
5b. Carboxymethylation of multisquamatin....... 15
6a. SDS-PAGE of multisquamatin.................. 16
6b. Molecular weight of multisquamatin.......... 17
7. CNBr digestion of multisquamatin............. 19
8. Clostripain digestion of multisquamatin. ... 20
iv
Abstract
Disintegrins are potent inhibitors of integrin function such
as platelet aggregation mediated by linking to fibrinogen.
The RGD (Arg-Gly-Asp) sequence and cysteine-rich motif are
conserved in the members of the disintegrin superfamily.
Multisquamatin was purified from Echis multisquamatus snake
venom. The four-step purification was performed on HPLC. By
using 16.5% SDS-PAGE gel, the molecular weights of multis
quamatin under reducing and non-reducing condition were
determined as 5.4 kDa and 6.8 kDa respectively. For deter
mining the C-terminal sequence of multisquamatin, cleavage
and digestion by CNBr and clostripain had been undertaken.
According to the HPLC profiles from digestion, there are a
number of minor peaks of unexplained origin affect the major
peaks which are to be used for sequence analysis.
v
Introduction
Integrins, cell-surface receptors, play an important
role in cell adhesion, differentiation, cell migration
during embryogenesis, thrombosis, and phagocytosis (1-6).
The superfamily of integrins are identified as heterodimers.
The & subunit of integrin contains a large extracellular
domain including four repeats of a forty amino acid cyste
ine-rich motif and a short cytoplasmic domain (1). The four
cysteine-rich motifs are conserved in most & subunits of the
integrin superfamily. The a subunits of integrins are com
posed of one heavy and one light chain linked by disulfide
bonds. There are more extensive homologies in C-terminal
region and Ca+2/Mg+2 binding motif is general in extracellu
lar domain of the a chains. The extent of this homology is
40-50% at the amino acid level (2) . The RGD or similar
sequence is common in ligands that bind to members of the
integrin superfamily. Different cell-surface receptors
mediate different responses in cell-cell/cell-matrix inter
action. The ligands include fibronectin, laminin, vitronec
tin, fibrinogen, von Willebrand factor, entactin, C3bi, and
collagen (1-6). Most of them bind integrins mediated
through RGD sequence to achieve cell adhesion or other
regulatory mechanism. Not only the RGD sequence but also
the conformation of these proteins influences the interac
1
tion. The other sequences of these proteins may alter the
conformation through binding to expose the RGD motif.
Recently, a number of low molecular weight peptides
have been isolated. These peptides are disulfide-rich and
are called disintegrins. In the early studies, it was found
that these peptides could inhibit the initiation of platelet
aggregation. Further result from the amino acid composition
and sequence of trigramin, one of these peptides, indicated
that these peptides have an RGD sequence. Through their RGD
sequence, these peptides bind to a cell-surface receptor on
platelets, glycoprotein(GP) Ilb/IIIa, with higher affinity
(nmol/1) than fibrinogen (7,14,15). The peptides which act
as inhibitors of physiological ligands such as fibrinogen
are named "disintegrins11. The potent inhibitors of integrin
functions have been purified from venoms of the Viperidae
and Crotalidae families (7,10-25).
The disintegrins are classified into three subfamilies:
a short group containing 48-49 amino acids (echistatin and
eristostatin)? a medium group that contains 70-7 3 amino
acids (trigramin, albolabrin, elegantin, agkistrostatin,
applagin, batroxostatin, flavoridin, and rhodostomin), and
a long group with 83-84 residues (bitistatins) (7,11).
Disintegrins have several characters in common such as short
2
half-life, acting as nontoxic inhibitors, RGD-loop conforma
tion, and cysteine-rich motif (7-10). The low molecular
weight disintegrins are considered to be derived from
multifunctional proteins such as the high molecular mass
hemorrhagic metalloprotease HR1B from Trimesurus
flavoviridis venom (8,23). The disintegrins inhibit platelet
aggregation induced by ADP, thrombin, collagen and platelet
activating factors, but can not influence the second
messengers such as cyclic AMP and Ca+2 (10,15). The
inhibitory mechanism of disintegrin is mediated via binding
to the receptors of the cell surface. These antagonists of
integrin receptors can be used as antithrombotic drugs.
In the past few years, different laboratories have
tried to find more species of disintegrins. Echis multisqua-
matus venom has been analyzed for its components (26,27),
but there is no report discussing the presence of
disintegrins in this venom. The present work reports the
four-step purification and the sequence of a disintegrin
from the venom of Echis multisquamatus.
3
Experimental Procedure
Materials
Lyophilized venom was purchased from Latoxan, Rosans,
France through its outlet, Accurate Chemical and Scientific
Corp., Westbury, New York. HPLC-grade TFA (triflouroacetic
acid) and BCA (Bicinchoninic acid) protein assay reagents
were purchased from Pierce, Redford, Tennessee. HPLC-grade
solvents were purchased from VWR Scientific, Cerritos,
California. Iodoacetic acid was purchased from Sigma, St.
Louis, Missouri. IEF and SDS-PAGE assay reagents were pur
chased from Bio-Rad, Richmond, California. All other
reagents were analytical grade. Sequencing grade
endoprotease, clostripain, was purchased form Promaga,
Madison, Wisconsin.
Instruments
Hydrophobic and ion-exchange chromatography was
performed on a Perkin-Elmer 410 HPLC system equipped with an
LC-95 UV/Vis detector and an LC-100 integrator (Perkim
Elmer, Norwalk, Connecticut) and a FRAC-100 fraction
collector (Pharmacia Biotech, Piscataway, New Jersey).
Reverse-phase chromatography was performed on a Spectra-
Physics 8800 HPLC system equipped with a Spectra-Physics
8450 UV/Vis detector and a Spectra-Physics 4290 integrator
(Thermo Separation Products, San Dimas, California). SDS-
4
PAGE was performed on a Bio-Rad Mini-protein II Electro
phoresis Cell. Platelet aggregation inhibition was measured
with a platelet aggregometer (Helena Laboratories, Beaumont,
Texas).
Columns
Hydrophobic interaction chromatography (HIC) column,
PolyPropyl A ( 25*210 mm) was purchased from Poly LC,
Columbia, Maryland. Reverse-phase C18 218TP1022 (4.6*250 mm)
and 218TP54 (22*2 50 mm) column was Purchased from Vydac,
Hesperia, California. Ion-exchange Synchropak CM300 (10*250
mm) column was purchased from SynChrom, Lafayette, Indiana.
Purification of multisquamatin
one gram lyophilized venom was dissolved in 4.5 ml HIC
buffer A (0.1 M phosphate, 0.02% NaN3, 1.0 M ammonium
sulfate pH 6.8). the solution was centrifuged (8°C) for 30
min at 10,000 RPM. The separated supernatant was loaded onto
a HIC column. The flow rate was 5 ml/min and the gradient
was from 100% to 0% of 1.0 M ammonium sulphate over 9 0 min.
The active fractions (as measured by inhibition of human
platlet aggregation) were pooled and then loaded onto C18
preparatory reverse-phase column. The 8 0% acetonitrile (in
0.1% TFA) gradient was operated from 0% to 30% in 90 min and
then from 30% to 100% in 10 min. The flow rate was 7 ml/min.
5
The active fractions from the first run were collected and
dried in vacou. This sample was redissolved in 0.1% TFA and
applied onto C18 preparatory reverse-phase column again by
using a similar acetonitrile gradient as the previous run.
The percentage of acetonitrile was from 10% to 30% in 80 min
then from 30% to 100% in 10 min. The pool of active
fractions after the second C18 reverse-phase column was dried
in vacuo and redissolved in CM3 00 buffer A (5mM phosphate,
pH 6.0) then purified by CM3 00 cation-exchange HPLC. A
sodium chloride gradient was used from 0.0 M to 0.5 M in 50
min and then from 0.5 M to 1.0 M in 15 min. The flow rate
was 2.4 ml/min. Aliquots of all fractions were assayed by
platelet aggregation inhibition test to identify the active
fractions.
Platelet aggregation inhibition assay
Human blood (36 ml) was drawn into 4 ml 0.1 M citrate
buffer (sodium citrate : citric acid = 6:4) and centrifuged
at 500 RPM for 2 0 min, 2 5°C. The supernatant, platelet-rich
plasma (PRP), was decanted into a clean plastic tube. The
platelet-poor plasma (PPP) was prepared from the remaining
blood by being further centrifuged at 10,000 RPM for 15 min,
2 5°C. PRP (3 00 fil) was mixed with PPP (2 00 /xl) and incubated
for 3 min in aggregometer at 37°C. 5 ( i 1 of each HPLC column
fraction was added into the reference cell at the start of
6
reaction then ADP (5 /xM final) was added after 1 min to
initiate aggregation. Inhibition was measured at the maximum
aggregation response.
Reduction and Carboxymethylation
The pool from ion-exchange chromatography was loaded
onto C18 analytical column again to desalt. The pure peptide
(~80 jLtg) was dissolved in 300 /xl of reaction buffer (5 parts
of 8 M guanidine-HCl and 1 part of 1 M Tris-HCl/4 mM EDTA,
pH 8.5) then 0.17 6 mg of dithiothreitol was added into the
solution for 1 hr at 25°C in the dark. After addition of 20
mg iodoacetic acid for 2 0 min at 25°C in the dark, the
caboxymethylated peptide was isolated by reverse-phase HPLC
as described above.
Cleavage with clostripain
The carboxymethylated peptide (30 /xg) was dried in
vacuo and redissolved in working buffer (25 mM Tris-HCl/ 1
mM CaCl2/ 1 mM EDTA, pH 8.0) followed by addition of
clostripain (peptide/ protease = 20/ 1) for 4-hr incubation
at 3 7°C. The reaction solution was loaded onto analytic C18
reverse-phase column to isolate the fragments.
Cleavage with CNBr
About 50 ug carboxymethylated peptide was redissolved
7
in 80% formic acid followed by addition of 530 mg CNBr at
25°C in the dark. After 24 hr, another 53 0 mg CNBr was added
for 12 hr. The complete digestion solution was loaded onto
analytic C18 reverse-phase column to isolate each fragment.
8
Results
Purification of multisquamatin
Multisquamatin was purified from the soluble portion
of the lyophilized crude venom of Echis multisquamatus by
four-step chromatography. In the early studies (28),
reverse-phase HPLC was the final step of the purification of
the disintegrins (11-13,15-19,25). Subsequently, cation-
exchange (CM 3 00) chromatography was added as the final
procedure (Figs. 1-4). Cation-exchange chromatography shows
that the pool from reverse-phase preparatory C18 HPLC still
contains several components (possibly isoforms of multisqua
matin) . Before multisquamatin was carboxymethylated, an
aliquot was desalted on C18 reverse-phase analytical HPLC
(Fig. 5a).
Reduction and carboxymethylation of multisquamatin
Carboxymethylated multisquamatin was eluted about 10
min earlier than intact multisquamatin from C18 RP-HPLC (Fig.
5b) . The small peak at about 60 min is uncompletely modified
multisquamatin.
Molecular weight of multisquamatin
Multisquamatin was loaded on 16.5% SDS-PAGE gel under
reducing or non-reducing condition. The bands were analyzed
for relative mobility using semi-logarithm profile (Fig. 6b) .
9
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Fig.l. Purification of multisquamatin by hydrophobic
interaction chromatography (HIC). Crude venom (lg) was
dissolved in 4.5 ml HIC buffer A (0.1 M phosphate, 0.02%
NaN3, 1.0 M ammonium sulfate pH 6.8) and loaded in Poly LC
hydrophobic interaction HPLC column. The column was
equilibrated by HIC buffer A and the flow rate was 5 ml/min.
The gradient of 1.0 M ammonium sulfate was from 100% to 0%
over 90 min. The peak of active fractions is marked by an
arrow.
10
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Time (min)
Fig. 2. Purification of multisquamatin by reverse-phase
chromatography (I) . The fractions from HIC column was loaded
onto Vydac C18 reverse-phase column. The 80% acetonitrile (
in 0.1% TFA) gradient was from 10% to 3 0% in 90 min then
from 3 0% to 100% in 10 min. The flow rate was 7 ml/min. The
peak of active fractions is labeled by an arrow.
11
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50 60 70 80 90 100 1 10
Time (min)
Fig. 3. Purification of multisquamatin by reverse-phase
chromatography (II). Fractions from the first reverse-phase
chromatography were pooled and dried in vacuo. Then this
sample was loaded onto C18 reverse-phase column. The acetoni
trile gradient was from 10% to 30% in 80 min then from 30%
to 100% in 10 min. The flow rate was 7 ml/min. The peak of
active fractions is labeled by an arrow.
12
0.8
0.8
E 0.6
c
0.6 "a
0.4
0.2
0.0
0.0
10 20 30 40 50 60 70 0
Time (min)
Fig. 4. Purification of multisquamatin by CM300 cation-
exchange chromatography. The fractions from reverse-phase
were dried in vacuo and redissolved in 5mM phosphate buffer,
pH 6.0. This sample was applied onto Synchrom CM300 cation-
exchange column. The column was equilibrated with 5mM phos
phate and then a sodium chloride gradient was applied from
0.0 M to 0.5 M over 50 min and from 0.5 M to 1.0 M in 15
min. The flow rate was 2.4 ml/min. The peak of active
fractions that were pooled is marked by an arrow.
13
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0 20 40 60 8 0
Time (min)
1 0 0 1 2 0 1 4 0
Fig. 5a. Multisquamatin on C18 analytic column. Before
carboxymethylation, multisquamatin was reloaded onto Vydac
C18 reverse-phase analytic column. The column was
equilibrated by 0.1% TFA and 80% acetonitrile ( 98 : 2 )
then 80% acetonitrile gradient was from 2% to 100% in 98
min. The flow rate was 0.8 ml/min. The major peak eluted at
about 55 min (27% acetonitrile). The peak fractions
collected are labeled by an arrow.
14
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Time (min)
- 0
120 140
Fig. 5b. Carboxymethlation of multisquamatin. After
carboxymethylation reaction, the solution was applied onto
Vydac C18 reverse-phase analytic column for desalting. The
same gradient was used as above. Carboxymethylated
multisquamatin (peak 1) was eluted at about 45 min (17%
acetonitrile) . Unmodified multisquamatin (peak 2) was eluted
at about 60 min. The large early peak (peak 3) is salt.
15
m
e.
s.
Fig. 6a. SDS-PAGE of multisquamatin. Lanes 1 and 4 are
low molecular weight markers: 31kDa, carbonic anhydrase;
20.4/19.7kDa, soybean trypsin inhibitor doublet; 16.9kDa,
horse heart myoglobin; 14.4kda, lysozyme; S.lkda, myoglobin
CNBr fragment 1 (FI); 6.2kDa, myoglobin (F2); and 2.5kDa,
myoglobin (F3) . Lanes 2 and 3 are 1 fig of nonreduced and
reduced multisquamatin, respectively. The higher molecular
weight proteins in lane 3 are artefacts since the protein in
lane 2, from which the lane 3 material was derived, is
homogeneous. The gel was stained by silver stain procedure.
16
100
Q
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w
ra
o 6.S
0 )
O 5.4.
2
'reduced
0.2 0.3 0.4 0.9 0.6 0.7 0.5
Relative Mobility
0.8 1.0
Fig. 6b. Molecular weight of multisquamatin. From SDS-
PAGE, the molecular weight v.s. relative mobility of low
molecular markers is used to generate the standard curve
shown in the profile. 0.76 and 0.70 are the relative
mobilities of reduced and nonreduced multisquamatin
respectively. The molecular weights of reduced and
nonreduced multisquamatin are 5.4 and 6.8 kDa, respectively.
17
The molecular weight of multisquamatin was analyzed under
reducing and non-reducing conditions and shown to be 5.4 kDa
and 6.8 kDa, respectively.
Cleavage by protease and CNBr
The chemical digestion and the cleavage by CNBr were
undertaken as described above. In addition to the undigested
peak (carboxymethylated multisquamatin), there were four
peaks from the CNBr digestion in two major groups and two
minor peaks in one. There was not base-line resolution
between those peaks (Fig. 7) . It was therefore difficult
with these samples to do sequencing analysis.
Cleavage by clostripain yielded a number of peptides
(Fig. 8) . Good base line profiles were obtained following C18
reverse-phase HPLC of working buffer only and clostripain
without peptide. After digestion of the reduced carboxyme
thylated disintegrin was completed, the solution was loaded
onto C18 analytical column. Chromatography revealed a number
of unusual minor peaks which affected the real peaks of
peptides fragments from digestion (Fig. 8) . After several
trials, there was no solution to the problem of the large
numbers of fragments obtained and the complex HPLC profiles.
The clostripain digestion has not been successful.
18
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Fig. 7. CNBr digestion of multisquamatin. Arrows 1, 2
and 3 identify the two major groups and one minor group,
respectively. The gradient, flow rate and column were as
same as described in Fig. 5a. In comparison with Fig. 5b,
there is no peak at about 45 min ( carboxymethylated
multisquamatin), so it appears that the CNBr digestion was
complete. Peak 4 is salt from working buffer.
19
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Fig. 8. Clostripain digestion of multisquamatin. This
profile shows the pattern of clostripain digestion of multi
squamatin. There five major peaks and many minor peaks close
to the background noise level. The gradient, flow rate and
column are as same as described in Fig. 5a. In comparison
with Fig. 5b, peak 1 or 6 is undigested carboxymethylated
multisquamatin possibly. The three early peaks are salt from
working buffer.
20
The amino acid composition of multisquamatin
The pure peptide was sent for composition analysis
using different conditions for the cysteine and methionine
analysis (Table 1).
21
Table I: Amino acid composition of multisquamatin.
Amino Acid Multisquamatin
No. of Residues
Alanine 1
Arginine 4
Aspartic acid 6
*Cysteine 81
Glycine 3
Glutamic acid 2
Histidine 1
Isoleucine 1
Leucine 1
Lysine 5
*Methionine 1
Phenylalanine 1
Proline 3
Serine 1
Theronine 3
Tyrosine 1
Valine 0
TOTAL 42
* Determined separately as cysteic acid and methionine
sulfone after performic acid oxidation
22
Discussion
Purification of multisquamatin
Reverse-phase HPLC did not lead to multisquamatin loss
of activity. From the platelet inhibition assay, the active
pool from the C18 column still inhibited aggregation (data
not shown).
Regarding the experience of digestion, the peptide was
not so pure as we expected. Cation-exchange chromatography
was added into the procedure. The second run of reverse-
phase chromatography show only one major peak. But when
major peak was loaded into CM3 00 column, several peaks were
observed. Through the platelet inhibition assay, there was
one peak which was very active. The other fractions still
inhibited aggregation at low levels. It was believed that
these fractions were isoforms.
Clostripain and CNBr digestion
The reason for digestion is so that we can determine
the carboxyl-terminal sequence of multisquamatin. The
reasons for the choice of clostripain are that this enzyme
can cut the amino terminal of Arg specifically and that
clostripain which is very hydrophobic can be separated from
the fragments of the digestion by C18 reverse-phase HPLC.
23
The profile of base line showed that the clostripain
was eluted vary late in the gradient, even the self-diges
tion was not so obvious. The peptide was digested by clo
stripain after four-hour incubation, but the peaks isolated
by reverse-phase chromatography were several times more than
we expected. There was not baseline resolution between the
major and minor peaks. Thus, we feel that something unusual
happened during the digestion.
From these experiments, it is a nice test to make sure
that the clostripain is pure enough for the digestion by
running the peptidase on the SDS-PAGE or HPLC.
Cleavage by CNBr seemed better than clostripain, but
there was no pure peak from each fragment for sequencing.
The whole digestion experiments should be improved to get
complete digestion and better resolution during chromatogra-
phy.
Sequence of multisquamatin
Because of the results of digestion, the fragments were
not able to be used for sequencing analysis. The sequence of
some fragments which did not give base line resolution on
the HPLC profile has been attempted. However, the sequence
was not interpretable from these analyses (data not shown),
24
because several amino acids were frequently identified at a
single residue location. After repeating the digestion
experiments several times, there was still no good fraction
which showed base line resolution on HPLC profile for
sequencing.
There are several ways for us to try sequencing multi”
squamatin. The carboxypeptidase A, B or Y can be used for the
C-terminal sequencing. From the known sequencing of
multisquamatin from N-terminal, the DNA probe could be
synthesized to hybridize the gene of multisqumatin in the
DNA library. Then the sequences of DNA and peptide of
multisquamatin can be obtained.
25
References
1. Hynes, R.O.: Integrins: A Family of Cell Surface Recep
tors., Cell, 48, 549, 1987
2. Ruoslahti, E., and Pierschbacher, M.D.: New
Perspectives in Cell Adhesion: RGD and Integrins.,
Science, 238, 491, 1987
3. Springer, T.A.: Adhesion receptors of the immune
system., Nature, 346, 425, 1990
4. Phillips, D.R., Charo, I.F., and Scaborough, R.M. :
GPIIb-IIIa: The Responsive Integrin., Cell, 65, 359,
1991
5. Yamada, K.M. : Adhesion Recognition Sequences. , J. Biol.
Chem., 266, 12809, 1991
6. D'Souza, S.E., Ginsberg, M.H., and Plow, E.F.:
Arginyl-glycyl-aspartic acid (RGD): a cell adhesion
motiff., TIBS, 16 July, 246, 1991
7. Gould, R.J., Polokoff, M.A., Friedman, P.A., Huang,
T.F., and Holt, J.C.: Disintegrins: A family of
Integrin Inhibitory Protein from Viper Venoms.,
P.S.E.B.M., 195, 168, 1990
8. Blobel, C.P. and White, J.M.: Structure, function and
evolutionary relationship of proteins containing a
disintegrin domain., Curr. Opin. in Cell Biol., 4, 760,
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Asset Metadata

Creator Ho, Chung-Kang (author)

Core Title Purification and sequence study of platelet aggregation inhibitor from snake venom

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Markland, Francis S., Jr. (committee chair), Danenberg, Kathleen D. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c20-316884

Unique identifier UC11258069

Identifier EP41848.pdf (filename),usctheses-c20-316884 (legacy record id)

Legacy Identifier EP41848.pdf

Dmrecord 316884

Document Type Thesis

Rights Ho, Chung-Kang

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA