isolation, partial purification, and characterization of … · isolation, partial purification,...
TRANSCRIPT
ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION OF
GOAT ALPHA-ONE PROTEASE INHIBITOR
BY PARVEEN SALAHUDDIN
•PARTMENT OF BIOCHEMISrRY ^. N. MEDICAL COLLEGpf
ALI&AJlH MUSLIM UNIVER'^SITY ALIGARH (INDIA)
Date
Approved:-
M. Abnl Qadm, Supervisor
A Dissertation submitted in partial fulfilment of the requirements for tlie degree of A/aster of Philosophy in BunUcmistry in the
faculty of Medicine oj the /iligarn Musi; in Un.^'eiMty AUG AH,;
1986
ChRTIFIGATE
I cer t i fy t ha t the work presented in the following pages
has been carr ied out by Miss Perveen Salahuddin and t h a t i t i s
su i table for the award of M.Phil, degree in Biochemistry of the
Aligarh Muslim Universi ty, Aligerh.
/
(M. Abul Qasim) Lecturer in Biochemistry, Department of Biochemistry, J.N, Medical College, Aligarh Muslim Universi ty, Aligarh,
I I
In t h i s d i s s e r t a t i o n , i s o l a t i o n , p a r t i a l pur i f i ca t ion and
brief charac te r iza t ion of c - i - p r o t e a s e i n h i b i t o r from goat serum
i s described.
cx- i -proteese i nh ib i to r from goat serum was Isola ted by
subjecting i t to amrionium su l fa te f r ac t iona t ion , a f f in i ty chroma
tography and ion-exchange chrcMnatography. F i r s t a crude preparation
of the i nh ib i t o r was obtained by col lec t ing a 50-80/^ ammonium
sulfa te fract ion which contained most of the inhibi tory a c t i v i t y .
This f ract ion was passed through a Cibacron-blue i?epharose 48 column
equi l ibrated with .01 M sodium phosphate buffer, pH 6 .8 , Ihis s tep
removed almost a l l of the albumin end ce r t a in other prote ins v*iich
had an af f in i ty for Cibecron blue. Finally the i nh ib i to r was pur i
fied by ion-exchange column diromatography on a i^i=At-cellulose
column ec^ i l ib ra ted with 0.005 M sodium phosphate ixiffer, pH 6 .5 .
The bound proteins were eluted using stepwise e lu t ion with 0,005 M
sodium phosi:*ate buffer containing 0.07 M NaCl and 0 ,1 M NaCl respec
t i v e l y . Two peeks were obtained, c< -1-pro tease i nh ib i to r was
located mainly in the second peak. The purity of th i s preparation
was checked by polyacrylamide ge l e lec t rophores i s . One major and
two ve:cy f a in t bands were obtained, suggesting t h a t the i nh ib i to r
preparation was f a i r l y pure. bDS-polyacrylaroide gel e lect rophores is
gave two bands corresponding to molecular weight values of 67,000
and 56,000. The inh ib i to r thus i so la ted showed strong inhibi tory
ac t iv i ty against t ryps in i r r e s p e c t i v e of the fac t whether synthet ic
i l l
substrate like BANA or BAPNA was used or a protein substrate like
casein was used. Further it was found that one molecule of inhibi
tor inhibited nearly three molecajles of trypsin indicating that the
inhibitor has multiple binding sites. The influence of two proteins
namely bovine serum albumin and porcine gamma globulin on the inhi
bitory activity of the inhibitor was investigated at different
protein concentration. Interestingly, albumin caused marked
increase in the inhibitor/ activity of inhibitor whereas gamma
globulin had no effect. The reason for the enhancements of inhibi
tory activity is not clear at present.
iv
I an greatly indebted to my supervisor, Dr* M. AbuX Qasin
for helping me at each and every ciucial stage of my research work.
I am also thankful to Prof, Salahudiih, Ghainnan, for provi
ding me fac i l i t i e s for my research wozic.
I have no words to thank to my senior colleagues Mr« Saad
Tayyab, Mrs. Kinsshtar Salman, Mrs. Ra^eedunnisa, Dr. Sudhir Kimar
Agaxwal and Mr. Tauseef Saeed Khan.
I also appreciate the cooperation of my colleagues
Miss, Renu Tyagi, A^ss Najma M i , Miss Manjeet Kair, Miss Nausheen
Haleem Khan, Mr. Kri^nan Hajela, Mr. Mir A^zaffar, Mr. Khalid
Majid Fazli, Miss Sadhana Shazma and Miss. Seeraa Hassan.
I am also thankful to non-teaching staff Mr. Behzad Khan
and Mr. Mohd. Nasir.
I also acknowledge to Indian Council of Medical Research for
assistance as Junior Research Fellowship to st»jdy this project.
. . . . (Parveen Salahuddin) Aiigarn May, 1986.
Pag«
ABSTRACT III
ACaCNOWLEDGEJlENTS V
DEDICATIC2J VI
LIST OF TABLES IX
LIST OF FIGURES X
LIST OP ABBREVIATICM3S XII
I, INTRODUCTION 2
II, EXPERIMENTAL %$
A, Materials, 25
B, Methods, 29
1) Meastir«nent of pH^ 29
2) O o t i c a l measurement 29
3) Determlnatlcm of p r o t e i n coiic«ntrati<»». 29
4) Ion-exchange chr<Mnatography on DEAE- 31
c e l l u l o s e .
5) Preparat l<»i of a f f i n i t y coltam 32
6) Polyacry lamide g e l e l e c t r o p h o r e s i s , 33
7) Soditsn do-decy l s u l p h a t e e l e c t r o p h o r e s i s ^ 3
8) I s o l a t i o n and p u r i f i c a t i c m of g o a t o C - 1 - 37
P r o t e a s e i n h i b i t o r ,
i ) ^imonium s u l f a t e f r a c t i o n a t i o n , 37
i i ) A f f i n i t y chrcwnatography on Cibacrcai 38 b l u e Sepharose-4B.
i l l ) DEAE c e l l u l o s e chrc»natography ,jj
v i i
Page
9) Measurement of Inhibitory activity of 39
C7^-1-protease inhibitor.
i) Using Casein as substrate, 39
ii) Using BAWJA as substrate. 40
iii) Using BANA as substrate, 41
III. RESULTS AND DISCUSSION 43
1) Isolation and p\irification of goatc<:-l- 43
protease inhibitor,
2) PAGE 55
3) SDS-PAGE 55
4) Influence of serton albumin and gamma 58
globulin on inhibitory activity.
5) Effect of inhibitor concentration on its 58
inhibitory activity,
IV, REFERENCES 59
V, BIOGRAPHY 64
VI, LIST OP PRESENTATIONS 65
viii
LIST OP TABLES
Paqm
Table I / nlnoacld and carbohydrate conqpositicms of hvBian« 5
rat, isotise and rabbit (Fast and Slow cooEponent)
oc-1-protease and mouse ccmtrapsin.
Table II Half time of association of different proteinases 19
with htanan plasma inhibitors.
Table III Coinparison of physico-ch«aical properties of 21
htsnan, rabbit, rheusus intmlcey and rat <^-1-protease
inhibitor.
Table IV List of chemicals used in this study, 25
Table V Isolati<»j and purification ofc^ -1-protease inhi- 47
bitor fran goat plasma. Inhibitory activity was
assayed using BAPNA as std:>strate.
Table VI Isolation and purification ofc^-1-protease inhi- 48
bitor from goat plasma. Inhibitory activity was
assayed using SANA as sijbstrate.
Table VII IsolaticHi and ptirificaticai of <3 -1-protease inhi- 49
bitor frc»n goat plasma. Inhibitory activity was
assayed using Casein as substsate.
Table VIII Molecular weight and relative iwability for diff- 53
erent proteins in sodium do-decyl sulfate polyac-
rylamide gel electrophoresis.
ix
I4»7 OF FX6URES
Fig, 1 fmiRO acid mmtemnoB of Inasaiicx -l-protcafttt inhibitor. 4
Fig, 2 StrtKsttirs of oligosacchari(!te coi^»oii«nt of oc -l«|>rot- 10 ease i sh ib i tor .
f i g , 3 Hypothetical structtara of inhibitor. 14
Fig. 4 Cos^^arisioii of inhibitory ac t iv i ty i» aiff«racit 42
nasRalat goat, s h a ^ buffalo «id rsdobit.
Fig, 5 Affinity chromatography &a Cib«srao bliMi sephartMMi 44
-4B and locatiem of inhibitory ac t iv i ty in diff«»
rtmt fracti<»i9 tasiag BAH4A, BmA «8id Casein aa
substrates.
Fig, 6 Iaa««xch«ige chromatography on DE E .. ce l lu lose 45
«ttJd location *f inhibitory ac t iv i ty in different
fractions ttsing«BM^A, BMA mad Casein as subst
rates*
Fig, 7 PolyiK;ryl«Ride gel eleetrophcnresis of oC-l-ilirotease, 50
inhibi tor .
Fig,8(A) Sodium do-diKsyl sulphate poly«^ryl«Ride ge l e l s e - 51
tz^::^hore8is of cX-l-protease inhibitor*
Fig,8(B) Sodium do-decyl sulphate polywsrylisnide gel e l e c - 52
trophoresis of >x-l-f>rot«aee inhibitc»r and marker
proteins*
Fig, 9 Plot of logrsthims of molecular iraight verstui 54
re la t ive mobility.
Fig. 10 Sffact of 8SA and IgC5 on aZbunto a^l«t«*oC- l - 56
protease inhibitor .
Fig. 11 Effect of diff«r«nt inhibitor coiic«atrati«ai cm 57
i t s inhibitory a c t i v i t y .
3ei
LIST GF ABBREVIAnONS
AT-III
e\ -l-Achy
c< -i-Ac
< -.2-AP
BAHA
BAPNA
C.-Inh.
DEAE-cellulose
HCl
NeCl
NaOH
A - i - P I
I - ^ - I
RBC
SDS
TEMED
TGfe
Tris
Antlthrorabin-III
(^ - i-Antichymotrypsin
oC-i-Anticoliagenas#
oc -2-Antiplaan in
c/:-N-Benzoylargine napthylaroide
(^-N-Bpnzoyl arginine-DJUp-nitroermilide HZl
Cj^-irihibitor,
Diethyl aminoethyi c e l l u l o s e
Hydrochloric acid
Sodijirj chlor ide
Sddium hydroxide
c^ - i - P r o t e a s e i n h i b i t o r
In t e r -oC- i - an t i t ryps in i r t i i b i t o r
Red blood corpuscles
Sodium dodecyl s u l f a t e .
N,N,N» ,N»-tetraraethylene athy lenedianine
Trichloroacet ic acid
Tris (hydroxy raethyl)methyl amine.
x i i
I,NTR(yXJgTIQN
i^rotease i nh ib i to r s ere present in p lants micro-organians
(Sves and mcmmals. Their presence was f i r s t demonstrated by Fertni
ouu jt ernoss i beck in 1894. In mamtnals protease inh ib i to r s are
believed to perform several important functions such as connective
t i s s u e turnover, coagulation, f i b r i n o l y s i s , complement f ixa t ion e t c .
They are found in various c e l l s as well as in d i f fe ren t body f lu ids
including plasma. In plasme t h e i r concentrat ion i s about lO,o of
the t o t a l plasma protein , i t cons t i t u t e s the l a rges t group of plasma
protein a f te r albumin and gamii a qlobul in . Though various types of
protease i n h i b i t o r s have been recognized the exact physiological
ro le of most of the protease i n h i b i t o r s i s unknown. The reason i s
t h i s t h a t not many pathological condit ions a re known where j u s t one
of the protease inh ib i to r i s missirwf or de f i c i en t end secondly most
of the i nh ib i to r s inh ib i t several d i f fe ren t proteases . The physio
log ica l ro le in such cases can only be derived ind i rec t ly from the
k ine t i c s of inh ib i t ion of various proteases with the i n h i b i t o r .
In the following few pages a br ief desc r ip t ion of molecular
proper t ies of x - l - p r o t e a s e i nh ib i to r from human are presented.
Typ?$ 9f Pypt?^^? |nh4W9ys
Protease inhibitors are classified according to the type of
protease which they inhibi t . The four major classes of proteases
are as follows:-
1. Thiol proteases
2. Metallo proteases
3, Aspartic proteases
4, Serine proteases.
Inh ib i to rs for a l l the four c lasses of proteases are known.
For example A -1-protease i nh ib i t o r (^ - i - P I ) group and the i n t e r
/ - t r y p s i n inh ib i to r ( I - T C - I ) i n h i b i t s only serine proteases , x - 1 -
cysteine protease inh ib i to r ( ^ -1-C3*I) i n h i b i t s only cysteine pro te
ases \fchereas ?<- i -an t i -co l l agenase {.\~iAC) i n h i b i t s only collegeno-
l y t i c enzymes of the metalloenzyme c l a s s . In cont ras t t o these c lass
specif ic i n h i b i t o r s < -2-Mecrogiobulin (-^-2-M) i n h i b i t s members of
a l l the four protease c l a s s e s .
Jf various c lasses of protease i n h i b i t o r s , ' ^ 4 - P I i s the
best understood. I t s physiological ro le has been es tab l i shed . The
primary function of i nh ib i to r i s to control the turnover of lur^
connective t i s s u e proteins by inh ib i t ing the e l a s t a se enzyme. The
deficiency of t h i s enzyme causes increased turnover of lung connec
t ive t i s s u e causing the development of pulmonary emphysema, U>mtt
of the important protease i n h i b i t o r s which belong to t h i s c lass are
A- i -p ro tease i nh ib i t o r , Antithrombin I I I (AT- I i l ) , A -2-Antipiasmin
(c<-2-AP), .Tv-i-Antichymotrypsin ( ^ - i - A c h y ) end CI- inhib i tor
( Cl-Inh, ).
Primary St ruc ture ;
The primary s t ruc ture of human c;C-i«protease inh ib i to r has
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been sequenced by c l a s s i c a l sequencing techniques (Johnson and
Travis, 1978;TroVls e t a l , 1978; Shochat e t a i . , 1978; Garrel i
e t a l , , 1979) cloning and sequencing of c *»A for baboon end human
Ck - l- i^I (Keruchl and Chandra e t a l . , 1981).
Humantx -1-pro teese i n h i b i t o r i s a r e l a t i v e l y sraail and polar
prote in . Thus, i t eas i ly passes through extravoscular spaces
( C a r r e l l e t a l . , 1982), The amino acid composition of ^ - l - p r o t e a s e
inh ib i to r from various animal species , such a s , human (Travis , 1974),
rhesus monkey (Berninger e t a l . , 1976), r a t (Rol l and Glew, 1981),
mouse ( Takahara and ^inohara, 1982) r abb i t (Koj e t a l , , 1978) has
beef) determined. Ihe amino acid composition from these animals i s
sumriarized in Table I . All of them have s ingle polypeptide chain
end are glycoprotein in na ture . The human < -1«PI vdiich i s by far
the best studied of a l l the Inh ib i to r s contains 394 amino ac ids . I t
has glutamic acid a t i t s I^-teitninal and lysine a t G-terminai. I t
contains 12% carbohydrate by weight, cDNA s tudies reveal tha t ^ - 1 -
protease inh ib i to r do not have pro-peptide but has a 24 residue
hydrophobic or pre-s ignei peptide (Carlson and i,tenflo, 1981). The
i nh ib i t o r molecule has e s ingle r e r c t i v e centre methionine loccted
a t posi t ion 358 in the amino acid sequence. Acidic amino acid
residues are concentrated a t N-terminal and basic amino acids are
more abundant at G-terminal.
ex - l - p r o t e a s e i nh ib i to r lacks d i su l f ide bonds. Instead i t
has a single cyste lnyl residue which forms d i su l f ide bond to e i t he r
cysteine or g lu ta th ione . The reason for t h i s pecul iar bond i s s t i l l
6
a mystery (Jeppson et a l . , 1976). However, i t i s thought t h a t in
nat ive s t a t e ^ - i - p r o t e a s e i n h i b i t o r ex i s t s in reduced s t a t e having
free t h i o l groups which perhaps pro tec t X - l - p r o t e a s e i nh ib i t i on
for being oxidized by chemical o r b io log ica l oxidants (Travis and
Salveson, 1983),
Reactive Centre:
The reac t ive centre of CT^-l-protease i n h i b i t o r has been
debatable. Due to i t s o r i g i n a l name x - i - a n t i t r y p s i n , lysine and
arginine were thought t o be present a t i t s r eac t ive cen t r e . On
ace ty la t ion of lys ine and arginine by maleic anhydride (Heimberger
e t a l . , 1971), c r i t acon ic anhydride (Johnson, 1978) and acet ic anhyd
r ide (Fretz and Gan, 1978) the i n h i b i t o r a c t i v i t y was abolished.
On modifying with less bulky methyl group, inh ib i tory ac t iv i ty
was not l o s t (Busby and Gan, 1976). However, various s tudies on
chemical modification of methionine residue suggest t h a t t h i s residue
i s primarily involved in the i n t e r a c t i o n with t a r g e t pro tease .
.< - i - p r o t e a s e i n h i b i t o r i s capable of i nh ib i t i ng an e n t i r e
ser ine c lass although with d i f fe ren t r a t e s . The presence of mult iple
reac t ive cen t re was therefore suggested (Feste e t a l , , 1981; James,
1978; Cohen, 1979; Martodam, 1981). But l a t e r on i t was revealed
t h a t on d i s soc ia t ing the complex of o^- i -p ro tease i r tn ibi tor to any
of these ser ine proteases i . e . t r yps in , chymotrypsin, plasmin or
e l as tase by nucleophiles , and the same peptide was cleaved in each
case
9
Third controversial debate bout the reactive eentre was
the position of the reactive c«itre methionine. Most of the workers
cited that methionine is located near N«.terminal (Panell et.al;
1974? Mor*i et.al., 1978f Crawford, 1973| Cohen et.al., 1978). This
ccmfusion arose because on coRiplex dissociation the cleaved peptide
binds throt^h hydrc hofoic forces to N-terminal. This jjeptide can
however, be dissociated frcrni .terminal by strong detergents like
sodium dodecyl sulfate ^ca^rell et*alf 1979f Morii et.al, 1979).
C^ analyzing the amino acid sequence of both nidified inhibitor and
the peptide fragment, the overlapping amino acid seqpience was obtained
(Travis and Johnscm^ 1978 ) thi» proving that reactive centre
methionine is located near Ct^rmixial.
Another question which was asked by workers (Del Hars et.al;
1979| Nakajima et.al., 1978f Mc Rae et.al., 1980) in this area is
that y an easily oxidizable group like methi(^ine is present at
the reactive centre. Apparently the presence of methionine at the
reactive centre appears to be disadvantageous, since this methionine
group is very prone to oxidation and thereby inactivating the inhi
bitor.
In fact, it has been found that replac^R^t of methionine by
valine at the reactive centre prodxices an alnwst equally effective
inhibitor which has the added advantage that it cannot be inactivated
by the various cocidissing agents which are produced in the cell.
Three explanations have be«n given sti fgesting the importance of
n«thi<»iine residue at the active sitet-
10
SIALIC ACID SIALIC ACID SIALIC ACID
GALACTOSE
GLcNAc
MANNOS
MANNOSE I
GLcNAc
GLc U Ac
GALACTOSE I
GLc NAc
ASPARANGINE
F i g . 2 . ( A ) Structure of ol igosaccharide component ofoC-i-protease I n h i b i t o r . ( Hodges e t . a l . , 1979.)
11
SIAL
MANNOSE-
GLcNAt
GUN Ac
ASPARANGINE
C ACID
GALACTOSE
GLc NAc
MAN NOSE
Fig.2 . (B) Structure of oligosaccharide component of (^C-l-protease i n h i b i t o r . ( Hodges e t . a l . , 1919,)
12
i) Rate of inactivaticm of neutrophil elastase Is faster when
methicmyl residue is present at its active centre?
ii) Methionine reactive centre provides lung tissue remodelling
to occur dxiring oxidatiem. Had there been any other amino
acid residue it would have been very difficult to remodel
the Itsig tissue.
iii) The process of phagocytosis requires an active participation
of proteolytic enzymes. Inactivaticm of these cmsBymes by
the inhibitor wDuld have deleterious effect on the whole
process. The control over the inhibitory activity of inhibi
tor is provided by simultaneous secretion of oxidants by
phagocytising cells which inactivate inhibitor in the variety
of phagocytizing cell. This process would be possible only
if methionine residues were present at reactive centre,
Caybffhydfftt ig
Several workers have studied the structtire of carbohydrate of
htananoC-l-protease inhibitor. Two tyxses of oligosaccharide units
are proposed to be attached at a total of four sites in the inhibi
tor molecule. The oligosaccharide structure was proposed by Hodges
«t.ai,(l979) (see Fig, 2), However, there is sc«ne dispute about
the type of oligosaccharides and the nuinber of attachment points to
the protein molecule. For example. Roll et.al.Cl978) have proposed
four different types of oligosaccharide units attached at 3-4 points
in the protein ttralecule. The oligosaccharide units are composed of
mannose, galactose, N-acetylglucoseamine and sialic acid.
13
Effect of Chemical and 3ioIo:jiceI Jxidents on .<-L^Protease Inhibitor
The presence of methionine residue at the reactive centre
generated an intense investigation of chemical and bioiogicei
oxidants effect on X-i-protease inhibitor (Johnson et al., 1978;
Laskowski et ai., 1979; Jannof, 1979; Travis, 1979; Cohen et si.,
1979). Jn oxidizing chemically with iM~chlorosuccinamide or N-bromo
succinamide the biological activity was abolished, aiological
systems generate oxidants by utilizing H2J2, CI" (Carp, 1978;
Matheson et al., 1976; Matheson et al., 1979, 1980; Clerk et al.,
1981) and myeloperoxidase. Ihe biological oxididants thus generated
also inactivate the inhibitor.
Secondary Structure
The secondary structure of cZ-l-PI has been studied by
Jirgenson (1977) by C.D. spectral measurement. His result suggests
the presence of 30 --< -helix, 40; P> -pleated structure and 30/o
random coil. Thus the protein contains significant amount of secon
dary structure. In the absence of disulfide bond in this protein
the major stabilizing forces for the nctive structure are believed
to be hydrophobic and electrostatic interactions.
Teritarv Structure
Molecular organization of oC-i-protease inhibitor reveals it
to be highly ordered structure. u>berman et al. (1984) have proposed
a three dimensional structure of modified inhibitor through crystal-
14
Met358
394
Z342
P i g . 3 . (a) The reac t ive cent re of oc- i -an t i t ryps in i s exposed on residue 342, ( s i t e of the z-mutation.)
Ser359
Met
358
Fig.3*(l9) Cleavage by t a r g e t enzyn» r e l ea se s the loop to give the s table post complex form with met 358 seprated by 69 AOfrara ser ine 359.
IS
io^raphic s tud ies . The protein molecule has dimension of 67/. x
32 A^ X 32 A®. / c id ic and basic amino acids are present ot oppo
s i t e ends of e i i i p s o i d e l molecule, thus making molecule polar in
character . This highly ordered s t ruc tu re primarily serves to fix
the reac t ive centre on the exposed s i t e ,
Carrel l et e l . (1985) hypothesized t h a t in modified i nh io i -
to r the s t ruc ture of reac t ive s i t e i s 'orung with methionine 35B dt
one end of the molecule separated by 69 A^ from serine 559 at the
other (see Fig. 3 ) . in the in t ac t i nh ib i t o r methionine 358 must
be plucked out from a P - sheet to give s t rained loop ( see Fig. 3)
joining with ser ine 359. Thus the cleaved i nh ib i t o r is thermodyna-
miceily s table and the i n t a c t i n h i b i t o r i s me t e s t a b l e , which expic^ins
the d i f f i cu l ty in c r y s t a l l i z i n g the native >:-1-protease inh io i to r .
This view of molf^cule with an exposed react ive centre s u i t s with
the observation such as , vu lnerab i l i ty of a reac t ive centre to
chemiccl and b io logica l oxidants .
Role of Carbohydrate
Recently (i-eter et a l , , 1985} revealed the fac t , tha t the
function of carbohydrate moiety i s to s t a b i l i z e the native conforma
t ion of-^' -1-protease i nh ib i t o r . This was p rac t i ca l ly demonstrcted
by these workers tha t the half l i f e of unglycosylated -^^-i-protecce
inh ib i to r i s shorter in comparison to glycosylated < - i -pro tecse
i nh ib i t o r . The protein synthesized in yeast or :. Coli
(Rosenberg e t a l . , 1984; Hil l e t a l . , 1984) by DNA-recombinant
16
technolo^ is devoid of carbohydrate moiety, -ach an inhibitor
nnolecul^ is likely to be cleared rapidly from the blood circuletion.
Standard Mechanism
Interaction between protein end protease inhibitor follows
the standard mechanism. The general criterion for the standard
mechanism ere the following (Travis et ai, 1983).
i) Rate of association between an enzyme and inhibitor should
be faft.
2) Rate of dissociation of enzyme and inhibitor complex should
be slow.
The x-ray data (i-askowski, 1980; Robert et ai., 1972)
revealed that the geometry of an active site should be optimal i.e.
it should act as a good substrate. Now the question arises what
makes an inhibitor act as an inhibitor rather than act as a good
substrate. In terms of structural analysis this problem has not
been solved but kinetically it can be explained as follows
The Kcat/Km value for the hydrolysis of the peptide bond in
the inhioitor following the standard mechanism is very high but the
individual value of Kcat and Km alone is very low ( Laskowski, i980J.
Therefore, peptide bond hydrolysis is very slow. This hydrolysis
of peptide bond is not irreversible but rather reversible. Hence,
an equilibrium near jnity is formed. Consider the general standard
17
mechenism.
I + 'i > EI^ > C >£!-• -rh + I*
K-i
where t. is th€- protease and I and I ere virgin and modified inhioi-
tors respectively and C is the stable intermediate complex. The
stable complex C can be formed from either virgin or modified inhi
bitors. 3ut the apparent rate of association of modified inhioitor
with c- nzyme is rather slow in comparison to virgin inhioitor.
Deviation from the ;:.tandard Mechanism;
o<-l-protease inhibitor follows the standard mechanism
partially. The rate of association between enzyme and inhibitor
is very fast and the resultant complex so formed contains one mole-
culdi of each enzyme and reactants, aut it deviates from the stand
ard mechanism in other aspects (Travis et al,, 1983).
1) Modified ^-l-proteese inhibitor is inactive and cannot
recombinc with protein. The inhibition mechanism of
^-i-proteese inhibitor obeys the following scheme:
K. K^ K^
L + i - > tL ^ I * >b + 1*
K-i
where I cannot reassociate with enzyme i.e. reaction mechanism is
not reversible; hence, modified inhibitor is inactive.
18
2) The strength of the in t e rac t ion i s so strong, therefore i t
has t o be covalently s t ab i l i zed .
E f f e c t 9 f Ui 9He ffp ^^yym o^- j -Fyp^^g?? ^nh4^3,^y
Pulmonary emphysema i s associated with low level of c i r cu l a
t ing iA -1-protease i nh ib i t o r . On the other hand c i g a r e t t e smoking
increase the r i sk fac tor of emphysema. Cigaret te smoke a t t r a c t s
neut rophi ls in lung which in turn r e l ea se b io logica l oxidants , and
thereby, oxidize the <-X: - i - p r o t e a s e i nh ib i t o r . The r a t e of i n t e r
action of oxidized c< - i - p r o t e a s e inh ib i to r i s 3CX)0 times slower
than na t ive i nh ib i to r and therefore i t f a i l s to control the e las tase
a c t i v i t y ,
Another group of workers (Beith e t a l . 1985) suggested that
smoke does not oxidize methionine as already claimed by several
workers. Bather a compound present in c i g a r e t t e modifies the basic
amino acids of^^ -1 -PI . This a l t e r s the nat ive conformation, which
in turn reduces the r a t e of i n t e r ac t ion of enzyme and inh ib i to r ,
int^y^gtion 9f^~i-i^irqt???e ;nh4'JU9y wj,t!) knV^ Pr9^?^s$
In terac t ion of -X - i - p r o t e a s e inh ib i to r with serine proreases
has been studied in human and in mouse ( Takahara and oinohara, i9S3j.
In terac t ion of human X -1-protease inh ib i to r with d i f fe ren t
ser ine protease such as pancreatic and neutrophil e las tase (bchwic.
e t a l . , 1966; Jannof e t a l . , 1972; daugh and Travis, 1976, Trypsin,
u O +» •H
(0
(0
c
(A (0 c
+> o M
a o c o
•H • P •0 o o (0 (0 <D
'H O
(1>
I
ID
to
(4 O
O « n>
M O
o
I (0 (D C
a, e
^5
I •H C ,-4 0) (0 (
i (0
6i^
I
£ C
a
19
3 I
(0 « »-4 «) UJ-f*
5
in
o -4 » N • -4
1
1
1
1
1
o o -< X »n •
CO
t
1
0*
3 X •
o X o
+
1
1
«n~ q -4 X cn •
<N
i
t
ID
X -H CM
in
o
CO
00
o
8
tf)
X
m
•o CM
tf> CO
>o • o
M 0. 1 ^ 1 <0 JC
a
1
>» x: o < i -1 i «0 JC
a
CM • N
s u (D =! CM i <0 x: sx
CM
fO CO en
MD 00
JC
o
CM
8 o CM
1
IS
c •H
1
1
8 o :4
r«-NO
a <
I CM
i ID
00
c o
•H
O to u +» c o 2
(0
o •H
t c •H
C o
• P to >
•H +» U c
20
chymotrypsin (SchiwicX e t*a l l 1976)* cathepsln G (Beith and Travis#
1930)I thrcmdsin (Matl^son et:«al««i976)« plasnin (Sharpio e t . a l . ' 1966;
Harcz et .al«# 1966)* acrosin (Frltas et«al .* 1970}* t i ssue kalllkrein
(Fritz et .a l« ' i969)« Factor Xa (£ l i l san at«ai.« 1962)« F4K;tor Xia
(Scott e t . al .» 1982). skin synovial collafenase (Ibkoro e t . a l * ' 1972)
an^ Urokinase (Harris et* al*« 1969) has l»een stu^ie^.
In pet i t ion to t h i s saicraliial serine proteai^ (Berfvist*
1963; i^icher* 1973; Sasaki* 1975) i s a lso inactivated tey^-l-pro-
tea^» intiD»itor. aut ti^e inactivatiem i t s e l f cannot c lar i fy the
physiological role ofc^^l'-protease inhi]»itor rather the rate of
ine^tivation i s of priaary importance* Meamiresaent of tlie rate of
aasociati(»i shows that the rate of in^:tivatioR i s fa s t e s t with
neutrojE^il e lastas^ (see Talnle ZZ) ssiffestinf that pr^ary function
ofc/-1-protease inhibitor Is to control td^ e l a s t o l y t i c ac t iv i ty .
"nrypsiii inhi)»itory capacity in mouse i s greater in coraperison
to humancT^-l-Pl due to ttie presence of two isofozm inhU^itors. One
of the isof orni i s ccmtrapsin which i s a)i«»nt i s man and t^e other
isoform iso<-l-antitrypsin ti^ich i s homologous to huaanoc^-l-
protease inhi]»itor. In mouse these two isoforms have different
inhibitory spectrum. Mouse ^-1 -protease inhi)»itcr inactivates
chymotrypsin* e l a s t a ^ and thrOBri»in« where as mcmse ccmtrapsin inhi
bited trypsin* plaanin* and trypsin-l ike protests from sub-maxillary
glands. Ibe dif ferent inhibitory spectrum of Uiese two isofax^jts
suffest t^at these two isoinhibitors d i f fer in their physiolofical
r o l e s .
(0
>.
o B 3
X)
C
§ x:
I u
u • •^ IB to V
f|
O 4*
«
C
o • H
CO
a o
CO
o
(2 S
•a ^^ O , o*
(A a> • 3 C M 119 " H ^ <i> Cx:
•f» • H 4 J jQ «
I '^ - I «
c 10 (0 e "H
8 •H
a 8
a
u
Q
D
Z
00
CM
in I
• • o fo
o
Q 2
Q
e 3 o >
O '2 ' "^ JQ4J0O <V < Crg a
0) (0
•ri U O «^ ^ t H - 4 "H O H ' *>
9 ®^ li o. o< «o
21
c
8
o B «0
4 * * 0
-« o t3 n
Q
2
a 2
§
*
I
o o
•H O
•H C
x: •H
H (0
3
o S
o (0
g 6 <0
to
c
g •2
^
i ^ _ 3 O N «in OB -*
o 4*
> o
•-« o
o.
o
«
a
&
o
• • • Q Q Q
• » • 2 2 2
K Q • •
•H o 2
^ 1 5 . ^
00
H I • - • >0 '(T
CO
^ 00 I
CM
4 *
M o •p «» >» x: u
u 4» u
O JO 2 C O -H -4 •H<H X3w4 X i fH Q) 4 ) O B <0 H «0 O
22
Genetics end Variants;
Human c^-1-protease i n h i b i t o r occurs in a t l e e s t twenty
d i f fe ren t forms in general population. This protein i s encoded by
two independent a l l e l e at a s ingle locus r e s j i t i n g in autosomal
codominant inher i t ance . The va r i an t s are c l a s s i f i ed according to
t h e i r e lec t rophore t ic mobil i ty . For excm^le, PiBB (where ^ • i s
protease i nh ib i to r ) i s horoozygote for anodal va r i an t s and txZi i s
hoiaozygote for cathodal var ian ts and PiMM represents the horoozygote
for normal a l l e l e . These two genetic var ian ts i . e , b and Z, c<use
decrease in X -1-protease i nh ib i t o r concentration in plasma. This
can be explained as fo l lows:-
in i, var ian ts mutation of glutamic acid occurs at amino acid
residue 264 to Val ( Carrel l e t a l , , 1976), whereas in Z. var ian ts
subs t i tu t ion occurs a t glutamic acid 342 to lysine (Jeppson, 1976).
These two giutonic acid in normal M-allele forms s a l t bridge and
s t a b i l i z e s rX- l -p ro tease i n h i b i t o r molecule. Subs t i tu t ion of these
two glutamic acid cause decrease in s t a b i l i t y which was also shown
by heat s t r e s s method. This increases the turnover of v>^-i-piotease
i nh ib i to r and thus resu l t ing in decrease in ^ ' - l»pro tease i nh ib i to r
concentration in plasma from iOl-130 mg/lOO ml to 15 m^/lOO ml.
Physical Properties;
Physical proper t ies of ^X- i -pro tea te i nh io i to r s of d i f f . r t .n t
mararals are t eoa l t t ed in Table 111,
23
Isolation of c:<-I-protease inhibitor has been performed by
several workers using a variety of techniques. Norm ally isolat ion
of (X - i -protease inhibitor i s performed in the following s teps: -
( A ) E^DOval of Albunin
(a) Salt Fractionation
( Z) Ion-exchange Chrotaatography.
( A ) fieffi9Yg4 9 l AA^fflJ^n
Since albumin and o< - l -protease inhibitor have seme molecular
weights and i soe lec tr ic points» therefore, they caiuiot be separated
by gel f i l t r a t i o n or by ion exchange chromatography. However, they
can be separated in two wayst-
( i ) Affinity QiroffiatoaraDhv on Cibacron»blue SeDharQse-4B
It binds albumin through bil irubin binding s i t e and to those
proteins having dlnucleotide fold but not cx-i-proteese inhibitor
from plasma.
(2) Affinity O ffflgtMr^p^Y gn ffgn A- ep^gfpgg 4P
It binds (j< -1-protease inhibitor and most of the glyco
proteins but not albumin.
Albumin depleted plasma, isolated from previous step i s
subjected to sa l t fractionation. The inhibitor fraction i s used
for further purification.
24
(C) Ign E?^c^^nq» (;^r9ii|i^^oqraphY
Fiirther pur i f ica t ion can be carr ied out by ion-exchange chro
matography on DEAE-celiulose or DEAE-sephadex using l i n e a r gradient .
S i t e s of o^ . l - p r o t e a s e Inh ib i to r Svnthes is t -
The s i t e of i nh ib i to r biosynthesis has been studied by
using three techniques:
i ) Radio-labelled amino acid techniouei -
By t h i s technique c< -1-pro tease i n h i b i t o r synthesis was
detected in macrophages, monocyte and lymphocyte (Wilson e t a l . ,
1981).
2} |mipyp9flv9y^?geot g"d p$p9XJld?g^ St^l^l^nq ^?ghr^J^qMgg:~
By these two techniques synthesis of ex -1-pro tease inh ib i
tor i s found to occur in kupffer c e l l s , b i l i a r y epithelium - i s l e t of
Langerhans, i n t e s t i n a l epithelium, macrophages, neutrophi ls and
hepatocytes (Eriksson e t a 1. , 1981; Geobes, 1982, Ray e t a l . , 1977).
Recently Franklin and Baumann (1985) discovered t h a t in
mouse species (M.crota l i ) (^ -1 -pro tease inh ib i to r expresses i t s
mRNA in kidney ra ther than in l i v e r which i s then secreted in ur ine .
The physiological ro le of o^ -1 -p ro tease inh ib i to r so synthesized
i s not c lear a t present , however, i t i s capable of inh ib i t ing
t rypsin and chymotrypsin.
25
A* MataertaXs
The chemicals an€ ot lwr mater ia l s use^ in ^ I s study are
suiranar i ze i here •
TABUB IV
L i s t of chemical used in t i l l s s tudy.
S.NO.
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
1 0 .
1 1 .
1 2 .
1 3 .
1 4 .
1 5 .
Chemica l s
A c e t i c a c i d
Acetone
Acrylamide
Amldo schwao^tz
Ammonium p e r s u l f a t e
Ammoniion s u l f a t e
Annmoniuro sulphamate
BAPMA
BAKA
Blue i:textran-200
Bovine serum albumin
Brcmiophenol b l u e
Br<»ilne
Calcium c h l o r i d e
Chloroform
S p e c i f i c a t i o n
AR
lA
X«R
LR
LR
LR
AR
Lot
Lot
Lot
Lot
LR
LR
LR
LR
No.
No.
No.
No.
119 C-
98 C-
1388
,19 C-(
-045
-055
3115
Source
BDFL I n d i a .
BDH* Ind l a .
M}H« England.
E.Merck* Germany,
Mitu in<? l a .
- do -
E.Merck* Germany
Sigma Chero.Co./
USA.
do -
Pharmacia f i n e .
Slfraa Che.Co«
USA.
BDH« Enf land.
BDH# I n d i a .
Sarabhal* Ind l a .
BDH# Ind l a
16. c* -Chymotryps Inofen^ M t N0.986-317P Sigma. CheiB.Co,9SA»
26
S*HO« Chemical
17 • CiJsacron-blue
18* Cooraassle b r i l l i a n t blue
19, Copper s u l f a t e
2 0. Dia lyzer tubing
2 1 . Dichlorod iinethyl-s i l a n e
S p e c i f i c a t i o n
AR
LR
LR
1/100* diameter
LR
2 2 , D i e t h y l ©nino e t h y l c e l l u l o s e
2 3 . Disodium hyelrogen phosphate
2 4 . Ep ich lo rohydr in
2 5 . E thano l
2 6 . Glycero l
2 7 . Glycine
2 8 . Hydrochlor ic ac id
2 9 . linmunoglobulin Type-G
30. Lithium s u l f a t e
3 1 . ^- raexxaptoethanol
32. «k - l -naph ty l - emine 'el i h y d r o g e n c h l o r i i e
3 3 . N-N*-methylene b i s a c r y l ^nide
34. N , N # N ' # N ' - t - t e t r a methylene^laraine
35 . Orthophosphor ic ac id
36 . Ovalbtnain
3 7 . pH paper
Lot NO.59C-0243
Source
Sipaa Chem.Co.,USA .
- do -
sm» I n d i a .
Arthur H. Thcxnas' USA.
E.Merklc* Germany.
Sigma Chem.Co;USA.
AR
AR
Q i e m i c a l l y pure sample
LR
LR
AR
AR
LR
LR
LR
l&
LR
LR
Lot
i^H-:
/
HO.A-^503
L-10
BDH# I n d i a .
BDH# I n d i a .
Bloger^ India*
Serva* Germany.
BDH, I n d i a .
Sigma Chem.Co;USA.
E,Merck» Germany.
Ca lb iochem.Cal . USA,
BDH# England.
Koch-Light -Lab. England.
Fluka* AG* S w i t z e r l a n d .
BDH# I n d i a .
Sigma (%em.Co.USA.
E.ttexrek. Germanv.
27
S.NO.
38,
39 ,
4 0 .
41*
4 2 .
4 3 .
4 4 .
4 5 .
46 .
4 7 .
4 8 .
4 9 .
5 0 .
5 1 .
5 2 .
53*
54 .
5 5 .
5 6 .
5 7 .
5 8 .
59 .
QieraicaXs
Potassium ^ichronste
PotassitsQ hy^rofen* phtha la te . Potass i i ^ pemansanatis
Ril»oflavin
RiJaonuc l e a ^ -A
depharose-4B
Sodium azicie
sodium i»icaxliionats
Sodium carbcmate
sodium chXoride
sodiiaa djUiydrofen phospiiate
sodiura dodecyl mi l fate
sodium h/droxlde
sodium molykdate
soditua n i t r i t e
sodium potassium t a r -tara te
Sodiuoa tetralaorate
Sodiisn tung s t a t e
soya Isean tryps in inhi)>itor.
SDcrose
Sulfuric aci^
Tr lch loro-ace t i c acid
S f e c i f i c a t i o n
AR
AR
AR
LR
l o t . No. 184916
hB.
IM
LR
W.
LR
AR
LR
I«R
AR
X^
LR
AR
Iot.No.29C-8040
LR
LR
AR
Source
SDH, l ^ i a .
- do -
- do -
B.!4erck« Gez^any.
Sisco* Ind i a .
Pharm«M:ia fiiva Chem. tJpsala* SMsden.
PKi FblasHie* Poland,
BDH* India.
- do -
Glaxo-Lab.« Indla.
- do «
BDfe Englead.
3DH# India*
- do -
S. Merck# Germany.
QDH* Ii%3 i a .
Sara^a i« M. I i^ l a .
ffl3H« India .
Sigma chem.Co.f USA.
mM* India.
SaraMiai M.Chem.m India.
Rei<i e t De Haen» Germany.
28
S.No. Chanlcals Specification Source
60, Tris(hydroxy-m ethyl) LR methyl amine
61. Whatman f i l t e r paper No. 1
BDH, England.
W and R Balston Ltd., England.
All glass distilled water was used throughout the experi
ments.
29
pH measurements were made either on ELIOO U-iO pH-«ieter
using EXieo glass and calomel electrode or on EC digital pH-meter
model pH 5691 in conjunction with EC eonbination electrode. The
{^•Hoeters were routinely standardized with 0«05 M potassium hydro"*
gen-pthalate, pH 4,01 (at 23®c), in the acidic range and 0.01 M
sodium tetraborate buffer pH 9.2 (at 25^C) in the basic range.
Light absorption measurement in the visible range were made
on Carl Zeiss Jena spectrocolorimeter, Speckol.
Protein concentration was deteirmined by the method of JU ry
et al . (1951). Bovine sexum allMimin was used as standard.
The Folin-phenol reagent was prepared according to the method
rec(^mended by Folin and Ciocalteau (1927). In a round bottmn flask
50 grams of sodium tungstate, 12.5 grams of sodiwn molybdate* 25 ml
of 85^ orthof^os|:^oric acid and 50 ml of hydrochloric acid along
with ^ 0 ml of water were taken and refluxed for about ten hours.
Then 75 grans of litiiium sulfate, 25 ml of dist i l led water and few
drops of liquid bromine were added to i t . The mixture was heated
30
vdthout a condenser t o reiBov« excess of bromin3. After cooling the
br ight yellow coloured reagent was d i lu ted t o 300 ml by adding
d i s t i l l e d weter. Then the contents were f i l t e r e d and stored in an
amber colo^ired b o t t l e . This reagent was d i lu ted four times before
use .
(>3) Pyep^raU9n yf cPPP^y yggflgPlf
Ihe copper reagent was prepared using the following solu-
tionsj-
i ) 4% sodium carbonate (W/V)
i i ) 4^ sodium potassium t a r t a r a t e (w/v)
i i i ) 2% copper su l fa te (w/V)
Solutions ( i ) , ( i i ) and ( i i i ) were mixed in the r a t i o of
100 j l : l i n t h i s sequence to avoid p r ec ip i t a t i on . The br ight blue
coloured reagent was f i l t e r e d before u se .
(c ) Ii!,y9,gedarff.
To one ml of protein solution, five ml of freshly prepared
copper reagent was added and mixed well and was kept for ten minutes
at room temperature. To this was added one ml of diluted Folin-
phenol reagent. Colour was allowed to develop, for 30 minutes then
the colour intensity was read at 700 nm. aiank was also prepared in
a similar fashion except that in place of protein buffer was taken.
The protein concentration was computed from the standard plot of
bovine serum albumin.
31
Twenty grams of DEAE ce l l u lo se was f i r s t soaked over night
in d i s t i l l e d water. Then the f in 6 s wer e removed thoroughly by
decantat ion. After t h i s the r e s in was regenerated according to the
method recotnraended by Bailey (1967). F i r s t the res in was equ i l i b r
ated with 0 .1 M NaQH to bring the res in in sodium cycle. Then the
res in was washed on a bucchner funnel t i l l the pH of the s lurry was
cbout 8, After t h a t the res in was t rea ted with 0 .1 M Hydrochloric
acid. Again the res in was washed extensively with water t i l l i t
was free from acid and f ina l ly equi l ibra ted with the s t a r t i ng
kwffer.
Packing of column
Column was f i r s t washed with detergent and was then rinsed
with d i s t i l l e d water. Lower end of the column was f i t t e d with l a
tex tubing and the column was mounted v e r t i c a l l y in vibrant f ree
pos i t ion . Lower end of the tubing was closed by pinch cock and
glass wool was inserted a t the lower end and few glass beads were
added. One th i rd of the column was f i l l e d with operating buffer.
Moderately d i l u t e slurry was poured with the help of glass rod. when
the res in had s e t t l ed t o few centimetre the lower end of the tubing
was opened slowly to half of the operating flow r a t e . Then flow
r a t e was increased gradually t i l l i t was s l i gh t ly higher than
required.
32
Epichlorohydrin cross-blinked desuiphated sepharose 43 was
prepared according to the method of Porath e t a i , ( i 9 7 i ) . 20 grao
of Sephcrose-4B suspension was t rea ted with 18 ml of IM NaOH and
2 ml of epichlorohydrin e t room toaaperature in tho presence of
40 mg of sodium borohydride. The suspension was heated to 60^c with
s t i r r i n g on water bath and the react ion was stopped a f te r two hours*
The gel was washed extensively with d i s t i l l e d water on a laiucchner
funnel t i l l i t was free from a l k a l i ,
aiue-sepharose 43 was prepared by coupling cibacron blue to
epichlorohydrin cross linked sepharose 43 according to the procedure
described by Bohme e t a l . (1972). A solut ion of 200 mg of cibacron
blue in 50 ml of water was added dropwise with vigorous s t i r r i n g t o
a 10 gm suspension of epichlorohydrin cross- l inked sepharose-.43 in
350 ml of water a t 60*^C. After s t i r r i n g for 30 minutes, 45 j r im^of
sodium chloride was added and s t i r r i n g was continued for about one
hour. After t ha t the mixture was heated to SO^C and 4 grams of
sodium carbonate was added to the mixture and s t i r r i n g c3ntinued
for another two hours a t t h i s temperature. After coo l i i^ the
mixture was brought to room temperature and the gel wes washed
extensively with hot boil ing d i s t i l l e d water on a bucchner funnel.
I t was f ina l ly washed with the equ i l ib ra t ing buffer.
33
PolyacrylaBitelc gel electro^Bhoregls
Polyacrylaml^« f s l electrofA^orests of - l - p ro t ea se Inh ib i
t o r was car r ied out In 7»0% ecryleaaliie gel a t pH 8.3 according to
the procedure recoiamenBled by Davis (1964), Stock so lu t ions for
polyacrylsBRi«ie ge l e l ec t rophores i s %»re prepared as given Iselow.
l ) Stock Solutions
Solution A : -
Porty e igh t i n i l l i l i t r e of 1 N |4C1, 36.6 grams of t r i s
and 0,2 3 ml of 1EMED were taken. Tlie volxjane was maie up to 100 ml
with water and pH was adjusted t o 8.9»
Solution Bi -
Forty e i g h t m i l l i l i t r e of 1 N HCl, 5.98 graos of t r i s#
0.46 ral of TSMED weire taken in water . The pH was adjusted to pH 6,7
and the volume was made up to 100 ml with d i s t i l l e d water.
Solution C i -
Twenty e igh t grams of scrylami^e and 0,735 graaa of N-N*-
methyleiw bis-acrylamide %#ere dissolved in water anci f i na l volume
was m ^ e up to 100 ml.
Solution D : -
Ten grams of acrylamide an^ 2,5 grams of N-N*methylene
his acryl&ai&e were dissolved in water anA then the volume was made
up to 100 ml.
34
F i r s t of a l l gel tubes ( 0 . 5 x i i cm) were throughly washed
end dr ied . The tubes were s i l iconized with a 1% dichlorodimethyl
s i iane in chloroforra. After drying, the tubes were fixed ve r t i ca l ly
in a stand by inser t ing t h e i r lower end in to the well of rubber
stopper. Each tube was f i l l e d with 2.2 ml of small pore solut ion.
The surface of gel solut ion was careful ly layered with a few drops
of water with the help of a syringe t o get uniform s j r face . The
polymerization was complete within twenty miruite. After polymeri
zat ion water layer was removed with the help of w.hatman f i l t e r
paper. Then 0 .5 ml of large pore solut ion was t ransfer red in to each
gel tube and the surface was covered with a few drops of water
before keeping the tubes under f luorescent l i gh t for photppolymeriza-
t ion . After the la rge pore gel had polymerized the water layer was
again removed. The gel tubes were then fixed in e lec t rophore t ic
tank such t h a t the lower ends of the gel tubes were in contact with
the buffer in the lower chamber and the upper ends were in the upper
chamber. Now the assembly was connected t o the power supply^
Protein sample in glycerol was poured gently to each tube. Then the
remaining space in the tube was f i l l e d with e lec t rophores i s buffer.
F ins l ly , the buffer was poured in the upper chamber such t h a t the
upper end of the gel tube i s completely dipped in e lect rophores is
buffer. After t h a t , few drops of 0.02% broroophenol blue dye was
mixed in the upper chamber. Electrophoresis was carr ied out a t 210
vo l t s with a current of 4 mA per tube. When bromophenol blue dye had
moved more than three fourth of the t o t a l gel length, the power
'.^
supply was turned off and the ge ls were removed from gel tubes by
squir t ing water between gel and the ge l tube with t h e help of a
syringe f i l l e d with a long needle. The ge ls were stained with IA>
amidoSchwartz (Stock solut ion G) solut ion for on© hour. Destaining
was done mechanically by shaking the gels with 7. (V/V) ace t ic acid.
Sodium dodecvl su l fa te DOlyacivlamide oel e lect roohores ia
SDS-PAQE was carr ied out according t o the method of i «eber
and Osborn (1969).
The small pore so lu t ion was prepared by mixing 7 ml of
stock solut ion C, 10 ml of gel buffer (0 .2 M sodium phosphate buffer,
pH 7.2 , containing 2X> SDS), 0 .5 ml of ammonium persul fe te (1.5^) and
0.02 ml of TEMED and 2 .5 ml of d i s t i l l e d water.
The solut ion mbcture was then, careful ly poured in s i l iconized
gel tubes end with the help of a syringe and then few drops of water
was layered cereful ly over the surface of t h i s so lu t ion . The solu
t ion was then allowed to polymerize a t room temperature for 30
minutes a f te r t ha t water was r^aoved by soaking : t with wiatman
f i l t e r paper.
(b) PreoareUon of protein sample
Ten milligrams of prote in in two ml of 0.2 M sodium phosphate
buffer containing ij> SDS and IX> f' -merceptoethanol was taken in e
3 6
t e s t tube and heated in a boi l ing water bath for 5 minutes. The
protein solut ion was then cooled to room t«nperature and a few
drops of bromophenol blue and 0.4 ml of glycerol were added. Thirty
to f i f ty mic ro l i t e r of protein sample containing nearly 60-100 ug
protein was then applied over the surface of small pore gel in each
tube. The tubes were then placed in e lec t rophores i s assembly in the
manner described above. The e lec t rophores is was performed using
0.2 M sodium phosphate buffer contcining 0 .2^ i^S, pH 7 .2 , as
e lec t rophores is buffer. A current of 8 mA/tube was passed t i l l the
dye front had migrated to a d i s tance of about 5 cm from the point of
sample appl ica t ion . After e lec t rophores is the gels were taken out
from the gel tubes and stained for protein by coomessie b r i l l i a n t
blue R-250.
( c ) Staining
The s taining of the pro te ins and desta ining of the gel was
performed with coomassie b r i l l i a n t blue R-250 by the method of
Fairbank et a l . (1971), The following solut ions were prepared for
stc'ining and destaining of ge l s .
i>olution A
I t contains 25/& (V/V) isopropyl alcohol , lD,o (V/V) ace t ic
acid end 0.05% (W/V) cjomas&ie b r i l l i a n t blue R-250.
Solution B
It contains 10^ (V/V) isopropyl alcohol, lO,o (V/V) acetic acid
and 0.05^ ( w/V) oommassie brilliant blue R-250.
37
Solution Cs
This solut ion contains 0.0028^ (W/V) cjomassie br i i i ian 'c
blue and iO;*5 (V/V) ace t ic acid .
With the above three solut ions s ta ining and desteining takes
place siimilteneously (Fairbank e t a l , , 1971). immediately a f te r
e lect rophores is each gel was dipped in about 25 ml of solution A
at room temperature for overnight . After t ha t the gels were t r ea ted
with solut ion 3 for a t o t a l period of 8 hours and f ina l ly ge ls were
t rea ted with solut ion C for 6 hours. The destaining process was
complete with another 5-6 washings w-ith 10;^ (V/V) ace t i c acid.
I so la t ion and pur i f i ca t ion of ^ - l - a n t i t r v p s i n from goat plasma
- / -1 -p ro t ea se i nh ib i to r was i so la ted using three techniques:
1) Ammonium su l fa te f r ac t iona t ion .
2) Affinity chromatography on Cibacron blue s.epharose-4B.
3) Ion exchange chromatography on DEAE-cellulose a t pH 6 . 5 .
For the i so l a t i on of ^ - l - p r o t e a s e i nh ib i t o r fresh samples
of blood were col lected from the local s laughter house in t he prese
nce of appropriate concentration of anticoacpjlant l ike tr i-sodium
c i t r a t e . R8C from the c i t r a t e d blood was removed by centr i fugat ion
at 4000 rpm for 10 minutes and the plasma thus^pto|p^^l;M»6S^fcde
^^ c/ V - ' s.'' .
38
50% saturated with awionijura s u l f a t e lay adding jneh^ul^ite amounts o£
s o l i d aBsnonium s u l f a t e . Ihe preci4pitati(»3i was allowed t o take place
o v e m i f h t at 4°C» The contents were then centrlfu^ad at 6000 rpai for
half an hour and thm p r e c i p i t a t e which contained very l i t t l e i n h i b i
tory a c t i v i t y was discarded and ^ e supernatant was l»r(»ight t o 80%
aRsnonium s u l f a t e saturation* Afain* i t was Icept overnight at 4°C,
Next day the s o l u t i o n was centr i fuged a t 12000 rpra f o r half an hour
in a r e f r i f e r a t e d c e n t r i f u f e . Prec ip i ta te so obtained was d i s so lved
in minimum volxime of O.Ol M sodium ^oapnatM buf fer i ^ 6«8 and was
dialyased e x t e n s i v e l y against the saeie buf fer .
(2) AEf i n i t v chromatography <Ma Cibacron blue ^pharo8e-4B
serum albumin and(^-l-*protease i n h i b i t o r have nearly stfoe
molecular weights and i s o e l e c t r i c points* Iherefore* the ir separa
t i o n i s d i f f i c u l t e i t h e r on g e l f i l t r a t i o n or on ion exchange. This
can however be separated on Cibacron blue a^pharose 4B* s ince t h i s
t r i a z i n e dye binds albumin* <:?c-l-antichymotrypsin# coagulat ion f a c
t o r s and to those prote ins having d inuc leot ide fold# l i k e dehydro
genases*
Itie prote in preparation obtained by 80% airanonitxn s u l f a t e
p r e c i p i t a t i o n (about 50 mf/40 ml) was applied on a cibacron blue
Se{^aros«-4B (5 cm x 7 cm) column equi l ibrated with O.Ol M sodium
phosphate buf fer pH 6.8* Column was operated a t the rate of 40 « l / h r .
Unbound prote ins were e luted In a s i n g l e p r o f i l e , Ant i trypt ic a c t i v i t y
was assayed in each fr^:ti(H}s and inh ib i tor r i c h f r a c t i o n s were pooled
for further pur i f i ca t ion*
39
(3) DEAE'-celluloae chrxaraatOTaiBhv
Fract ions r ich in antitry]»tic a c t i v i t y were d ia lyze*
a f a i n s t 0.005 M sodium phOFhate buffer/ pH 6.5 an«l applieei on
D£AE-ceIlulose column ( 7 cm x 1.66 cm ) . The coliamn was washed
with two l»ei volumes of t h i s buffer t o remove unbound ]»roteins.
Bound pro te ins were e luted by incorporatinf 0.07 M NaCl and O.lOO M
NaCl in b u f f e r . Anti trypt ic r i ch f rac t ions were pooled.
III.Maasurement of inhib i tory a c t i v i t y of " l -nrotease inh ib i tor
( A ) The inhib i tory a c t i v i t y of -1-protease i n h i b i t o r
was assayed accordinf to the methocH of Anson (1938) us inf case in
as subs tra te .
Tfen mi l l i frams of tryps in in 10 ml of 0. Oi M Tris-HCl
(jrti 7 .7 ) conta in inf O.Ol N caiciurn chlor ide was tftk;en» case in so lu
t ion was prepared by d i s s o l v i n f 2 §m of c a s e i n in 0,06 M sodium
phophate buf fer pH 7.7 in 100.0 ml.
To one ml of protein* 0.1 ml of tzrypsln was added an t h i s
was incubated a t 37°C for 2 0 minutes. Then 3 ml of 10% TCA was added
and the reac t ion mixture was afain incxibated a t 37'* C for one hour.
A white p r e c i p i t a t e was obtained which was f i l t e r e d o f f . Control
was prepared in s imi lar fashion also* except that c a s e i n was ad^ed
a f t e r stoppinf the reac t ion with 10?4 TCA. The f i t r a t e so obtained
from the sample and contro l were assayed f o r t h e i r hyerolyzed pro
duct by the method of Lowry e t . a l . # (1951) .
The inhibitory activity of CK-I-protease inhibitor towards
the S3^thetic stjbstrate was measured using BARN A and BANA,
(l) Assay of ex -1-protease inhibitor using BAPNA as si±>strate»
The inhibitory activity ofc<-t-protease inhitor using
B4PNA as substrate was assayed according to the method of
Waheed and Salahuddin (1975),
Ten milligrams of BAENA was dissolved in 0,15 ml of dimet
hyl sulfoxide and the voliaroe was made to 10 ml with 0,01 M
Tris-*iCl, (pH 8,0) . Trypsin (10 mg/10 ml) was dissolved in the
same buffer containing 0.01 M calcixan chloride.
To one ml of proteiir, 0,1 ml of trypsin was added and the
reaction mixture was incubated at 37* C for ten minutes, Th«i
erne ml of BAPNA was added to this reacticm mixture and was
incubated for five minutes, Th®r» the reaction was stopped with
one ml of 30% acetic acid, TRe colotir intensity was measured at
410 nm,
(ii) Assay of A-1-protease ii dJsitor using BANA as substratex
The inhibitory activity of -1-protease inhibitor was
measured according to ^je method of Martineck et,al. (1964)
with slight nradificaticm. Ten milligrams of BANA was dissolved
in 0,5 ml of dimethyl sulfoxide and the voltime was made to
10 ml with 0,05 M sodiian phosphate buffer pH 6,2. Ten milli
grams of trypsin was dissolved in 10 ml of 0,01 M Tris-HCl
buffer pH 8,0 containing lOmH of calcitim chloride.
41
"^ one n l of lnhiJ»itor« O.l »1 of trypsin was aided and
Inculiated for 30 minutes at 37 C« After addlnQ 0.5 naX of SANA i t
was afain lncid»ated for 30 nlnutaes at 37°b. fhe reaction was
stopped liy the addition of 0.5 n l of 4 N HCl. llhe ccmtrol was
prepared in the s ^ ^ manner* except that ttim mlistrate was added
after the addition of 4 Ij HC1«
Ihe ^Rcnint of 2-nap^ylaBii» fozswd during tkm hydrolysis
of SANA at pH 6 .3 was determined toy couplinf and dias^t lsat ion.
This was done hy addinf 0«S ml of maiiiam n i t r i t e (0*2^ w/v) to
t^e solut ion. Ihe contents were i^aken thoroughly and ex&ctly
after three minutes* one ml o£ aamxaiAjm sulj^^aate (0«5^/v) were
added. Exactly after three mJUnutes two ml of couplinf dye i . e .
N-l-Napthylethylene diamine dihydrochloride (0,<B^w/v) in etha-
nol was added, The colour was allowed to develop for about 40
minutes and the intensity oi v i o l e t blue colour was read at 540 rm
42
PROTEIN CONCE/MTPATION LY\ W 3
P i g . 4 . Comparison o^ Inhibi tory a c t i v i t y in d i f f e ren t mamals; goat C O ) ' sheep ( # ) ' >^uffalo ( ( J >' r a b b i t ( • ) .
Inhibi tory a c t i v i t y was assayed according to the method of Waheed & Salahuddin (1975J
43
Before s t a r t i ng these s tud ies , preliroinaiy experiments on
the concentration of -X^-i«protease i nh ib i to r from the sera of
various animais such as goat, buffalo, sheep and rabbi t were perfor
med in order to s e l e c t a source which contains highest concentration
of i n h i b i t o r . For inhibi tory ac t iv i ty meaairement from these sera
30-80% ammonium su l fa te f rac t ion was obtained and inhibi tory ac t iv i ty
was measured against t r yps in . The r e s u l t s are shown in Fig. 4 . I t
can be seen t h a t a l l sera contain inh ib i to ry a c t i v i t y . However,
there are considerable quan t i t a t i ve differences , whereas in tiie
CtiQ of rabb i t serum 2.74 mg prote in was required t o cause 50%
inh ib i t i on , the goat serum was several time more ef fec t ive and
only 0.4 mg of protein was requiired to causa 50;^ inh ib i t ion under
iden t i ca l condi t ions . In the case of buffalo and sheep 0.74 mg and
1.25 mg was required to cause 50!^ i nh ib i t i on . These preliminary
s tudies suggested t h e t of the four sera t r i e d highest concentrexion
of inh ib i to r was present in the esse of goat serum. For t h i s reason
goat serum was selected as the source of i n h i b i t o r in these s tud ies .
n . l§9igU<?n end ourWggtlOT Qi ^9?\ ^ "I'pygtttgs^ inhi^^tgf
The 50.80^ ammonium su l fa te f rac t ion obtained during ammonium
sulphate f rac t iona t ion of goat serum was subjected to a f f i n i t y
chromatography on Cibacron blue Sepharose 4B. This step was essent ia l
44
40 80 120 160 200
ELUTION VOLUME.tT\4
Pig,5, Affinity chrcmiatography of 80% ammonium sulphate fraction on Cibacron blue sepharose- 4B,
The column (5cm x 7 cm) was equilibrated with ,01 M sodiiBTi phosphate buffer, pH 6.8. About 30mg of 80% ammonium sulphate fraction was applied on the coltimn and the unbound protein was collected in 10ml fraction at a flow rate of 40ml per hour. Inhibitory activity was located in different fractions using BAPNA ( ^ ) , BANA ( • ) , and Casein ( f) ) as sxdbstrate.
46
since albumin, which i s the major plasma protein present, i s similar
to '^ -1-proteese inhibitor in molecular weight and electxo phoretic
rK>bllity and without prior removal of albumin the i so lat ion of the
inhibitor free from aliMmin contamination i s very d i f f i c u l t . The
aff inity column would bind albumin as well as a l l other proteins
which contain dinucleotide fold such as dehydrogenases, but not
oc - l -proteese inhibitor among other proteins. The aff inity coiuotfi
chromatography result i s shown in Fig, 5, Almost a l l of the inhibi
tory act iv i ty was accounted for by the unbound fractions. The bound
fractions (mostly albumin) which could be isolated by eluting the
column with 2 M potassium thiocyanate in buffer was found to be
virtually free from the inhibitory ac t iv i ty .
Finally cx- l -protease inhibitor was purified by ion exchange
chromatography on a DEAE-cellulose column \AAiich was previCMJsly equi
librated with 0.005 M sodium pdiosphate buffer, pH 6 .5 . About 36 mg
of protein in 40 ml of 0.005 N sodium phosphate buffer, pH 6.5, was
applied to the ion exchange colunff). The unbound proteins were remo
ved by washing the column with 0,005 M sodium phosphate buffer, pri
6 .5 . The bound proteins were eluted by stepwise incre&se in ionic
strength. Two peaks eluting at ionic strength 0.07 and 0.1 were
obtained ( see Fig. 6 ) . The protein fractions obtained frc»n 0I:>.£-
ce l lu lose column were also monitored for inhibitory act iv i ty against
trypsin using SAPNA, SANA and Casein as substrates ( see Tables V, VI
end VII respect ive ly) . Both the protein peaks showed signif icant
inhibitory ac t iv i ty . However, the inhibitory act ivi ty was compara
t ively more in the peak obtained at 0.1 M ionic s t r ^ g t h . Active
47
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50
P l f . 7 , Poly aery lamide f e l e l e c t r o p h o r e s i s of goat cx- i -protease Inh ib i tor .
E lec trophores i s was performed in Tr i s - f lye i i i e buffer pH 8.3* 7.0% polyacrylenaide g e l . About 60uf px^tein was applied on § e l tube and e l e c t r o p h o r e s i s was perforraeg f o r ^ o u t 2 hour« with anodic current of 4 inA/tube. itie g e l s were stained wit^ cooraassie b r i l l i a n t blue and dest-ained with 1% a c e t i c ac id .
51
F i g . 8 , ( A ) Sodium io^ecyl sulphate polyacrylamlde gel e lec t rophores i s of goat JC-1-protease i n h i b i t o r .
Electrophoresis was performed using 0,2M sodium phosphate buffer pH 7,4 containinq 0, iJi SDS on lO)i poiyacrylaniide g e l . About lOO g containing 0,1% -mercaptoethnol wcs performed for about 3 hours with an anodic current of 8 raVtube. Itie ge ls were stained with coamassie hXne and destained with 10% ace t ic ac id .
52
Fig«8» ( B ) Soaivan oLodecyl s t i lphate p o l y a c r y l a n t d e g e l e l e c t x o p h o r e s i s of (A) Goat "^^-i-protease i n h i b i t o r , (B) Ovalbumin. (C) <^-Chymotrypsinogen (D) Soya bean t r y p s i n i n h i b i t o r (E) Ribonuclease (F) Bovine ^srum albumin.
E l e c t r o p h o r e s i s was performed us ing 0,2M sodium phosphate b u f f e r pH 7,4 c o n t a i n i n g 0,1^4 SDS on lOjS poly aery lam ide g e l . About 70 g c o n t a i n i n f 0.1% -mercap toe thno l was performed f o r about 3 hours with an anodic c u r r e n t of 8 mA/tube, Ihe g e l s were s t a i n e d wi th coamassie b lue and d e s t a i n e d witli 10% a c e t i c a c i d .
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02 04 OS OS
RELATIVE MOBILITY —*
Fi«,'9;. Plot •£ Mm •«lta»« of iMjrIwr pmtmimm iwrstas Xofrsthln 0i mol««til«r iraifht.
Th0 »«r)c«r protoiiui w«r« (1) Bovln* ••ran «lbuRii» M«Mt.69»000l. (2) Chralbumin (M.Mt«4S.000} . (3) • Chymotrypmifk9^^ti (M,Wt.25,500) . (4) Soya !>••& trypsin, inhibitor (M,lft,21,500) . (5)Ribonucl«a»« (II.Wt.l3,6SD •
55
fractions un<«r thia peak were pooled*
m * Parity of g^-^-»rot«aee tohiiittort
The purity of the inhibitor preparation ol»taii)«l after
ion*exchanf« chromatofraphy was checXedl l y poXyeeryl«ilie f e l e l e c
trophoresis in Tris-f lycine lauffer* pH 8*3 (Davis* 1964 )• As shown
in Fif• 7# one major Itand ami two veiy fa int liants were obtained in
eXectrophoreatOfr«a. These resul t s suffest l^at ^ e inhibitor pre
paration was fa i r ly homofeneous on eharfe liasis*
2V. ao^ija #oAecyl sulfate Polvacryl«iide aal elaetroBhoreaist
SDS-polyacrylamKe f e l electroiritoresis of t^e inhUtoitor
an<l the marlcer proteins was performed in 0,2 M soiiuin phophate buf
f er f>a 7«2# accoriinf to the procedure of weiier ani Glrt»om (1969),i!tM
resu l t s of sDs-electrophoresis of marker proteins anal the inhJJkitor
are shown in Fif . 8. In the case of inhibitor two protein bands were
(Stained, "me relat ive mobility of SEDS-protein complex for different
marker proteins was plotted molecular weifht. A s trai fht line was
obtained <see Fi f . 9) which af ter l eas t squsrs analysis f i t s into
the followinf equation.
Lof M m -1.236 -¥ 5.16
The re lat ive mobility of th« inhibitor-SDs-complax was
calculated to be 0.21 and 0.27 which accordinf to above equation
cozresponds to molecular weifht values of 67#000 and 5d«000 xespec-
t i v e l y .
56
I N H 1 B I T 0 R \ B S A (MoUr rat io )
0-93 186 3 0 0 372 4-^48 558 6-^8
0-86 172 25 3 4 4'i 4-8 BSA OY I9G I INHIBITOR (MoUr yatio )
Fig,j_(y Effect of BSA ( # ) and Ig.G ( O ) on albtimin depleted -1 protease inhibitor.
The BSA was incubated with different concentration of inhibitor from 2,89xliy®M to LSxlO-^M) ( O ) and similaryly the inhibitor was incub^ed with different concentration of BSA and IgG from 3.7x10"^ to 3.0xlO-=M ( # ) and 9.4xlO-6M to 9,0xl0-6M ( O ) respectively. Inhibitory activity was assayed using BAPNA as substrate according to the method of Waheed & Salahuddin (1975) .
St
o
X
z u. o
0-5 152 2-54 357 4-59
MOLAH RATIO INHIBITOR ENZYME
?ig.ll, Sff«ct of inhibitor concentration oa its inhibitory acitivity.
EiisBYBio trypsin (4,2x10"' was incubated.with different ccmc^atration of inhibitor from (8,4xl0~"^ to 1,07x10 M> Inhibitory activity was assayed using BAPNA as a stibstrate according to the method of Waheed & Salahuddin (1975) ,
58
Influence of tvvo proteins namely serum albuinln and gamma glo
bulin on the inhibitory activity of the inhibitor was studied at
different canc^itrations of these two proteins as well as the inhi
bitor. Ihe results are shown in Fig.10 * Qaania globulin had no
effect on the inhibitory ectivity of the ir^ibitor at least up to
five fold molar excess over inhibitor. Under the same conditions
bovine serum albumin cajsed percent inhibition to increase from 118
to 200. In fact there were almost a linear increase in inhibitory
activity upto a molar ratio of 4«8. Interestingly, vi en serum albu
min concentration was maintained constant and the inhibitor concen
tration was increased, a decrease In percent inhibition takes place.
Both of these results suggest that serum albumin increases 'ttie
inhibitory activity of the inhibitor.
Effect of different concentration of irUiibitor as shown in
Fig. 11 on inhibitory activity suggest that inhibitory activity
increases as inhibitor concentration i s increased. However, at
higher inhibitor concentration (molar ratio 3) inhibitory activity
remains constant suggesting that 3 molecules of enzyme had completely
inactivated one molecule of oc -1-protease inhibitor. This would
mean thct inhibitor has multiple binding s i t e s .
5f
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$4
BIOGRAPHY
Parveen SaXahuddin was born in Azaiagaih on Novamber 17,
1961. She passed High School Examination in 1977 from Aligaxh
Muslim University, Aligarh. She received her B.Sc. (Hons.) degree
in Chemistry in 1981 and M.Sc. degre in Biochemistry in 1984. She
was enrolled as Ph.D. student on 17th December 1984 in the
Department of Biochemistry, Jawaharlal Nehru Medical College, Aligaih
Muslim University. She is a member of the Society of Biological
Chemists (India).