measured with o-phthalaldehyde by the method of ... ture were added to 1 ml of o-phthalaldehyde solution and ..... trast, modification of trypsin with either poly-.
Agric.
Biol.
Chern.,
53 (8),
2063-2071,
1989
2063
Functional Changes of Polyethylene Glycol-modified Serine Proteinase from Aspevgillus sojae and Interaction with a2-Macroglobulin Kiyoshi Takoi, Youhei Yamagata, Tasuku Nakajima and Eiji
Department of Agricultural Tohoku
University,
Sendai,
Ichishima*
Chemistry, Faculty oj Agriculture, 1-1, Tsutsumidori-Amamiyamachi,
Miyagi 981, Japan Received
The covalent
attachment
of activated
January
31, 1989
polyethylene
glycol2
(PEG2)
of 10,000
daltons
to non-
essential groups on a serine proteinase II (SepII) from Aspergillus sojae produced two modified preparations
(PEG2-SepII-S
PEG2-SepII-L antigenicity,
were about 170,000 and 280,000, respectively. The PEG2-SepII-S lost about 80 % of its while the PEG2-SepII-L completely lost its antigenicity. In comparison of kinetic
and PEG2-SepII-L).
The molecular
weights
of PEG2-SepII-S
and
parameters with SepII there was less than 40 %variation in Km, but the values ofArcat towards succinylL-leucyl-L-leucyl-L-valyl-L-tyrosine
4-methylcoumaryl-7-amide
(Suc-LLVY-MCA)
or succinyl-L-
alanyl-L-alanyl-L-valyl-L-alanine />-nitroanilide (Suc-AAVA-/>NA) decreased to about 70 % less than that of SepII. The modified preparations have about 20 %activity towards fibrin hydrolysis and a low affinity for a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), with a molecular weight of 23,000, while the preparations have high affinity for a low molecular weight microbial inhibitor, chymostatin. The stoichiometry of the reaction of a2-macroglobulin (a2M) with PEG2-SepIIS showed that PEG2-SepII-L bound to a2Min a molar ratio of 1 : 1. No appreciable differences were observed in the pH stabilities of the modified enzymes and the native one at pH3.6, while the modified enzymeswere more stable than that of the native one at pHll.5. The two modified preparations were labile at 50°C, but the native enzymewas completely stable at 50°C.
The chemical modification with ylene glycol, (2-0-methoxypolyethylene
col)-4,6-dichloro-s-triazine
polyethgly-
(PEGJ or 2,4-bis-
(O-methoxypolyethylene glycol)-6-chloro-lstriazine (PEG2), of protein yields soluble adducts with several interesting properties. Abuchowski et al}~3) and Ashihara et al^ have shown that conjugation of PEG1? a nonimmunogenic polymer, to catalase or l-
asparaginase leads to a complete loss of their
antigenicity while retaining their enzymatic activity towards low molecular weight sub-
strates. The introduction of PEGXin sufficient amount to elastase5)
and streptokinase6)
ren-
ders these proteins non-immunogenic as judged by various criteria.
A serine proteinase (EC 3.4.21.14) from Aspergillus sojae, a microorganism used to make Japanese traditional fermented foods,
soy sauce and miso, is a DFP-sensitive enzyme with a molecular weight of about 23,000,7) and is not inactivated by tosyl-L-lysine or tosyl-L-
phenylalanine chloromethylketone (TLCKor TPCK).8) In a previous paper,9) we reported
* To whomreprint requests should be addressed. Abbreviations : a2M, a2-macroglobulin; AMC, 7-amino-4-methylcoumarin;
anti-SepII,
antiserum
to serine
pro-
teinase II; Boc-VLK-MCA; /-butoxycarbonyl-L-valyl-L-leucyl-L-lysyl-^methylcoumaryl-T-amide; PEGl9 (2-0methoxypolyethylene glycol)-4,6-dichloro-s-triazine or activated polyethylene glvco^; PEG2, 2,4-bis-(Omethoxylpolyethylene glycol)-6-chloro-5-triazine or activated polyethylene glycol2; SepII, serine proteinase II from Aspergillus sojae with a p/ of 5.2; SSI, Streptomyces subtilisin inhibitor; Suc-LLVY-MCA, succinyl-L-leucyl-L-leucyl-Lvalyl-L-tyrosyl-4-methylcoumaryl-7-amide.
2064
K. Takoi et al.
mo- Serine proteinase IIfrom Aspergillus sojae. SepII from
the heterogeneity of the electrophoretic
bilities, isoelectric points, and kinetic parameters of five forms of the serine proteinase
(SepI~SepV) from Aspergillus sojae. In the previous investigation we also reported an irreversible molecular conversion of the elec-
trophoretic multiple forms of the serine proteinase.^
The occurrence
of five forms of
serine proteinase from Aspergillus sojae was not attributed to self-digestion but to inactivation because of conformational changes. The substrate specificity of a major fraction
of the serine proteinase (SepII) was investigat-
ed with oxidized insulin A and B chains.10) Weshowed that SepII can hydrolyze the amide bond between lysine and 7-amino-4-methylcoumarin (AMC) in the Boc-VLK-MCA,n)
which (EC
is a sensitive 3.4.21.7).12)
substrate
The stoichiometry
of plasmin of the re-
action of a2Mwith the SepII showed that the
Aspergillus sojae was purified by the previously described method.9)
Assay for serine proteinase. Proteinase activity towards
milk casein at pH 7.5 was assayed by the method described.10) Fibrin solubilization was measured at pH 7.5. After precipitation of unreacted fibrin at pH 4.0, an increase in absorbance at 660nm was assayed with the Folin-Ciocalteau phenol reagent.1 5) Fluorometric activity towards Suc-LLVY-MCA,Boc-
VLK-MCA or the others was assayed as in our previous paper.13)
Kinetic parameter measurement. Kmand kcat were calcu-
lated from Lineweaver-Burk plots. Protein
concentration.
The protein
concentration
of
PEG2-SepII preparations was estimated by measuring the
amount of free amino groups with trinitrobenzene sulfonate16) after alkaline hydrolysis with 3.17m NaOH at 120°C for lOmin with bovine serum albumin as a standard.
A sensitive
amino terminal analysis was also used for
SepII bound in a molar ratio of about 2: 1.13) measurement of protein concentration. The terminal In this paper, we show that the modification amino groups of modified PEG2-SepII preparations were measured with o-phthalaldehyde by the method of of Sep-II from Aspergillus sojae with activated Roth.17) The mixture of 50 /zl of protein and 50 /il polyethylene glycol2 of molecular weight of of6.33n NaOHwas autoclaved at 120°C solution for 20min. After
10,000 greatly and completely diminished the
antigenic reactivity of the enzyme towards the antiserum while sparing its activity. Wealso aimed to obtain preparations that could be administered intravenously as a safe fibrinolytic drug. The highly modified PEG2-SepII-L, which dissolves fibrin, is almost certain to be non-immunogenic and may be of value for the treatment of venous blood clots. In this study, the interaction of a2M with slightly modified PEG2SepII-S was also investigated.
Suc-LLVY-MCA,
Boc-VLK-MCA,
a Hitachi
and the
spectrofluorometer
model F-3000.
tation and emission wavelengths respectively.
The exci-
were 340 and 455nm,
Modification of SepII with activated polyethylene glycol2 (PEG)2. The modification of SepII was done by the method of Abuchowski et al}~3) as follows: to a SepII solution (0.55mg/ml)
in 0.1m
borate
buffer,
pH 8.5, was added
0.662g of activated PEG2 with a molecular weight of 10,000. The mixture was incubated for 0~24h at 5°C and then fractionated by polyacrylamide gel electrophoresis at pH 9.4.18)
Materials and Methods Materials.
cooling, 250/A of 1.3n HC1 was added to the autoclaved mixture. Three samples of lOOjul of the neutralized mixture were added to 1 ml of o-phthalaldehyde solution and 2ml of H2O. The fluorescent intensity was measured with
Two active
bands
were extracted
from the
polyacrylamide gel with 0.02m acetate buffer, pH 6.5, by the method of Kobayashi et al.19) The polyethylene glycol modified preparations, PEG2-SepII-S and PEG2-SepII-L, were prepared by Superose 12 column chromatography of FPLC (Pharmacia Fine Chemicals Co.). Elution was done
others, and chymostatin were purchased from the Peptide Research Center, Ina, Mino-shi, Osaka, Japan. a2M from bovine plasma (Code No. 602442) was purchased from Boehringer Mannheim-Yamanouchi,Tokyo. Fibrin was with an increasing concentration gradient of salt (0.2m NaCl) in Na-phosphate buffer, pH 7.5. Two differently purchased from Nacalai Tesque, Inc., Osaka. 2,4-Bis-(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2, Lot 601423) (MW; 10,000) was purchased from Seikagaku Kogyo, Co., Ltd., Tokyo. Streptomyces subtilisin inhibitor (SSI)14) was a gift from Drs. S. Murao and K. Oda.
modified SepII preparations were prepared by changing the molar ratio of activated polyethylene glycol2 to amino groups in the SepII molecule (PEG/NH2: 6.0 and ll.0). The total amino groups in SepII molecule are 1 5, including 14 8-amino groups of lysine residues and 1 a-amino group
Functional Changes of PEG-modified Serine Proteinase
2065
of glycine.20)
and single
Polyaerylamide gel electrophoresis (PAGE). PAGEwas done using 7% polyacrylamide gel in Tris buffer, pH 9.4, at 20mA/plate at 4°C for 1.5hr; the protein was stained with Coomassie brilliant blue R dissolved in 50%
Interaction
methanol-9.5% acetic acid, and destained in 5% methanol-
Electron microscopic analysis. Electron microscopic anal-
9.5% acetic
acid and 7% acetic
acid as in a previous
Measurementof molecular weight. Molecular weights were measured by the gel filtration method with Superose 12 of FPLC. Elution was done with an increasing concentration phosphate aldolase
radial
from Aspergillus
wt.,
158,000),
trypsin (mol. wt., 20,400), wereused.
ovalbumin
(mol.
Difference spectrum. The difference spectrum was measured with a Shimadzu spectrophotometer UV-265. Measurement of antigenic activity. Antigenic activities were measured by the methods of immunoelectrophoresis
(a2M)
with
the
PEG-SepII-S
was
done as in our previous paper.13)
ysis was done with a Japan Electronics Instrument, JEM100b. A sample (0.25mg/ml) each of a2M and the a2MPEG2-SepII-S complex was negatively stained with 2% sodium phosphotungstate
(pH
7.5;
Nacalai
Tesque,
Inc.,
Osaka) on a carbon film (Veco 499 mesh, Ooken- Shoji Co.).
Results
wt., 43,000),
and RNase (mol. wt., 13,700),
ofa2M with the PEG2-SepII. The interaction
of a2-macroglobulin
gradient of salt (0.2m NaCl) in sodiumbuffer, pH 7.5. Standard marker proteins,
(mol.
immunodiffusion.21)
Figure
1 shows polyacrylamide
gel electro-
phoresis of the modified preparations, PEG2SepII-S and PEG2-SepII-L. The Rf values of SepII,
the
PEG2-SepII-S,
and
PEG2-SepII-
L were 0.5, 0.2 and 0.1, respectively. Further purified preparations were obtained by Superose 12 column chromatography of
Fig. 1. Course of PEG-modification of SepII from Aspergillus sojae on PAGE. Each sample was put on a 10% polyacrylamide gel and then electrophoresis was done at 20 mA/plate and 4°C for 1.5hr in Tris buffer, pH 9.4. Lane 1, SepII; lane 2, 3hr; lane 3, 6hr; lane4, 9hr; lane 5, 12hr lane 6, 24hr.
2066
K. Takoi et al
FPLC. The molecular weights of PEG2-SepIIS and PEG2-SepII-L were about 170,000 (210,0001 10,000) and 280,000 (>300,000~ 200,000), respectively. The amino groups of modified proteinase preparations, PEG2SepII-S and PEG2-SepII-L, substituted with
activated PEG2were about 6 and ll, respectively. Although the PEG2-SepII-S lost about 80% of its antigenic activity against anti-SepII serum by immunoelectrophoresis
and single
nitroanilide (/?NA), Suc-AAVY-/?NA, SucAAPF-/?NA, Suc-AAPL-/?NA, d-VLK-^NA, T0S-GPK-/7NA, Z-GPR-/7NA, Z-GPCit-/?NA
(Cit=citrulline), and Boc-LSTR-/?NA. The two PEG2-modified enzymes, PEG2-SepII-S
and PEG2-SepII-L, and native SepII showed similar substrate specificity towards synthetic
fluorogenic and chromogenic substrates (Table I). The kinetic
parameters
of PEG2-SepII-S
and PEG2-SepII-L towards Suc-LLVY-MCA radial immunodiffusion,21) when the antigenic and Suc-AAVY-/?NA at pH 7.5 are sumactivity of PEG2-SepII-L with the anti-SepII was measured, the antigenicity of PEG2-SepII- marized in Table II. Compared with native
L was found to be abolished completely (Fig. SepII and PEG2-SepII-S, there was less than 2). The homogeneity of the PEG2-SepII-S was 15% variation of Km, but the kcai values of confirmed by immunoelectrophoresis (Fig. 2). PEG2-SepII-S described to 40-46% less than
Two PEG2-modified enzymes can hydrolyze
the amido bond between an amino acid and 7-amino-4-methylcoumarin (AMC) in the peptidyl-MCA, Boc-VLK-MCA, Suc-AAPF-
MCA, and Boc-LSTR-MCA (Table I). Furthermore, the two PEG2-modified enzymes had some amidase activity on peptidyl-/?-
that of SepII. In PEG2-SepII-L, there were rather larger variations in the Km and kcat values than those of PEG2-SepII-S. The results indicate that the fibrinolysis activities of PEG2-SepII-S and PEG2-SepII-L are retained when insoluble fibrin was used as a substrate (Fig. 3). The retaining ofenzymatic
Fig. 2. Immunoelectrophoresis of PEG2-SepII-S and PEG-SepII-L. Each sample was placed in the well of an agarose plate and then electrophoresis was done at 20mA/plate in 0.38 m Tris-HCl buffer, pH 8.9, followed by immunodiffusion using anti-SepII serum for 3 days at 20°C. The immunoprecipitates were stained with Coomassie brilliant blue R after being washed with 0.9% NaCl. Lane 1 , PEG2-SepII-L;
lane 2, SepII;
lane
3, PEG2-SepII-S.
Functional Table I. Relative Activities PEG2-SepII-L towards
Changes of PEG-modified Serine Proteinase
Methylcoumaryl-7-Amides Peptidyl-/?-Nitroanilides
from Aspergillus
2067
of PEG2-SepII-S and Peptidyl-4(MCA) and (PNA)
The rates of hydrolysis of Suc-LLVY-MCAand Boo AAVA-/?NA are arbitrarily taken to be 100 for PEG2modified enzyme preparations and native one. S
ubstrate
Peptidyl-MCAsa Suc-LLVY-MCA Boc-VLK-MCA Suc-AAPF-MCA Boc-LSTR-MCA Peptidyl-/?NAsb Suc-AAVA-/?NA Suc-AAPF-/7NA Suc-AAPL-/7NA D-VLK-/7NA T0S-GPK-/7NA Z-GPR-/7NA Z-GPCit-/?NA B0C-LSTR-/7NA
Native
c TT2C SepII-S
1 00 53 1 3 8 1 00 79 1 5 1 6 50 9 49 37
c TT2_
1 00 42 14 8
SepII-L
1 00 42 1 3 1 0
1 00 8 1 1 3 23 57 11 47 45
Fig. 3. à" Time Dependence ofFibrin Hydrolysis SepII-S, PEG2-SepII-L, and SepII at pH 7.5. å , PEG2-SepII-S; V, PEG2-SepII-L; O, SepII.
by PEG2-
1 00 78 1 4 23 64 11 50 43
a Concentration of peptidyl-MCAs was 100/im. b Concentration of peptidyl-/?NAs was 50fiM. Boc, /-butoxycarbonyl; Sue, 7V-succinyl; Tos, A'-tosyl;
Z
iV-benzyloxycarbonyl.
Table II. Kinetic Parameters of Amidase Activities towards SUC-LLVY-MCA and SUC-AAVY-/7NA by PEG2-SepII-S and PEG2-SepII-L
Km .
Enzyme
.
,
kcat_1x
(mM) (sec
,
kcJKm _/
_1N
x) (mMxsec x)
Fig. 4. Effects of Concentration of a2M on the Enzymatic Activity of PEG2-SepII-S and PEG2-SepII-L. The reaction mixtures were incubated with various molar amounts of a2M in 50mMphosphate buffer, pH 7.5, at 30°C for lOmin, and then the residual activities of the reaction mixtures were measured with Suc-LLVY-MCA. å , PEG2-SepII-S;
Suc-LLVY-MCA Native
0.027
PEG2-SepII-S PEG2-SepII-L Suc-AAVA-/?NA
Native
0.
PEG2-SepII-S PEG2-SepII-L
0.35
0.031 0.038
15 0. 17 0. 18
0. 14 0. 10
3.9 1.8 1.3
1 3 5 3
26 11 7
activity might be caused by steric hindrance
between
the PEG2-SepII-S
or PEG2-SepII-L
and the insoluble substrate, fibrin. As shown in Fig. 4, the enzymatic activity of a2M-PEG2-SepII-S decreased proportionally with increasing amounts of a2M, while the
amidase activity for Suc-LLVY-MCA finally remained constant at 50% of the original
activity
V, PEG2-SepII-L;
O, SepII.
even in the presence of an excess of
a2M. On the other hand showing a high resistance
the PEG2-SepII-L, to a2M, probably
cannot readily form the complex with a2M, unlike the modified PEG2-SepII-S. sults show that the PEG2-SepII-S
These rewas stoi-
chiometricaly bound to a2Min a molar ratio of1:1.
The difference spectra in the region between
270 and 300nm are due to changes in the environments of the aromatic chromophores of the protein molecules.22'23* The time dependence of the difference spectrum at 294 nm of the
a2M-PEG2-SepII-S
complex
solution
versus a solution of the a2Mshowed positive
2068
K. Takoi
and sharp at or within
1min, but no peaks
were observed after 1 min (Fig. 5). The results
suggest a conformational change in the a2M molecule due to the binding to PEG2-SepII,
which is completed within 1 min. The conformational changes in the a2Mmolecule caused by the a2M-PEG2-SepII-S complex formation were identified from electron micrographs. It could be seen that there were two forms of the a2M-PEG2-SepII-S complex, an H-form and a football form as shown in our previous paper.13* The former may be a side face of the a2M-PEG2-SepII-S
complex,
and the
et
al
proteinaceous Streptomyces itor (SSI),14) the activities SepII-S, and PEG2-SepII-L
subtilisin inhibof SepII, PEG2decreased pro-
portionally with time, and 50%of the were activities of the three enzyme preparations abolished after 30min, spectively (Fig. 6).
60min,
and 80min,
re-
In Fig. 7A there were no appreciable differences in the pH stabilities of the PEG2-SepIIS, PEG2-SepII-L, and native SepII at pH 3.6, but the PEG2-SepII-S and -L were more stable than the native
one at pH ll.5
(Fig.
7B).
latter
About 50% of the activities of SepII, PEG2were abolished may be a perpendicular face of the a2M-PEG2- SepII-S, and PEG2-SepII-L after 3.2, 5, and 8min at pH ll.5, with the SepII-S complex in a ball. The activities of the PEG2-SepII-S and first-order rate constants, k, of 0.0034 sec"1, PEG2-SepII-L were equally small microbial inhibitor,
abolished by a chymostatin,24)
which was a powerful inhibitor of SepII with a Ki of 3 x 1(T8m.13) However the PEG2-SepII-
S and PEG2-SepII-L were incubated with the
Fig. 5. Time Dependence of the Difference Spectrum at 294nm on the Interaction of a2Mwith the PEG2-SepII-S.
Fig. 6. Course of Inhibition of the PEG2-SepII-S, PEG2SepII-S, PEG2-SepII-L, and SepII with a Proteinaceous Inhibitor, SSI, at pH 7.5 and 30°C å , PEG2-SepII-S; T, PEG2-SepII-L; O, SepII.
Fig. 7. Course on Acid Treatment at pH 3.6 (A) and Alkali Treatment at pH ll.5 PEG2-SepII-L, and SepII at 30°C..B, PEG2-SepII-S; å¼, PEG2-SepII-L; O, SepII.
(B) of PEG2-SepII-S,
Functional
Changes of PEG-modified Serine Proteinase
further
from Aspergillus
purified.
Also,
a certain
2069
amount of
enzyme appears not to be coupled by either of
these procedures.
This study showed that the modification of SepII with PEG2 produced PEG2-SepII-S and PEG2-SepII-L. We found that electrophoresis is useful for purification of PEG2-SepII-S and PEG2-SepII-L,
purification Fig. 8. Temperature Stabilities of PEG2-SepII-S, SepII-L, and SepII at pH 7.5. å , PEG2-SepII-S; V, PEG2-SepII-L; O, SepII.
PEG2-
as shown
in Fig.
1. Further
was done by chromatography on
FPLC. The modified PEG2-SepII-L showed a complete loss of its antigenicity against the anti-SepII antiserum by immunoelectrophoresis, while the PEG2-SepII-S showed the antigenic activity against the anti-SepII anti-
serum by immunoelectrophoresis, as shown in
0.0023 sec"1, and 0.0013 sec"1, respectively. Fig. 2. Loss of antigenicity of PEG2-SepII-S At 50°C, the activity of native SepII was was found to be about 80% by the single radial stable
at pH 7.5 for lOmin,
70% of the activities
while
60%
and
immunodiffusion
method.
of the PEG2-SepII-S and
Preparations of modified trypsin were proPEG2-SepII-L respectively were diminished duced by Abuchowski and Davis.3) The molar (Fig. 8). Furthermore, about 88% of the re- mass of trypsin was increased by about 15,000 sidual activity was observed with the PEG2- daltons. The preparation was designated PEGSepII-L at 40°C for lOmin, while more than trypsin (24%). The second preparation showed 96%of the initial activity was observed with a 59% loss of primary amino groups, which the same temperature and time by PEG2corresponds to the introduction of 8~9 PEG SepII-S
and complete
activity
was observed
with native Sep-II.
strands/trypsin was designated
molecule. The latter adduct PEG-trypsin (59%). Leonard
and Dellacheria27) reported that the molecular
weights
Discussion PEG2-modification of SepII The attachment of activated PEG to proteins is simple and occurs under mild conditions. With several proteins the reaction
yields homogeneousproducts, as seen by symmetrical peaksultracentrifuge, from a Sephadex column, in the analytical and in sucrose gradient
ultracentrifuge.1>2)
Only
ultracen-
trifugation is necessary to removeunattached
PEG. For purification of modified PEGjstreptokinase6) and PEG1-elastase,5) an ultrafiltration with XM-50 membrane to remove free activated PEG1 and chromatog-
raphy on Sephacryl S-20 were used. In contrast, modification of trypsin with either polyvinylpyrrolidone25)
or dextran26)
requires
a
greater numberof manipulations and often yields heterogeneous products which must be
of the various conjugates
of PEGr
hemoglobin were evaluated from the gel permeation chromatograms on AcA44 Ultrogel
and compared with those globular proteins. In this case, the elution profiles showed a single
but broad peak at a volume between the void volume (Mr>200,000) and that of aldolase (Mr> 158,000).
For measurement of the molecular weights of PEG2-SepII-S and PEG2-SepII-L, we also
used gel filtration with Superose 12 of FPLC compared with those of globular proteins described in the text. The elution volumes of the maximumof the peaks of the two modified enzymes correspond to those of a globular protein with molecular weights of nearly 170,000 and 280,000, respectively. Unfortunately the PEG2-SepII-S and PEG2SepII-L lost their ability to solubilize fibrin to a great extent as shown in Fig. 3. Although the
K. Takoi
2070
et al.
decreased fibrinolysis ability of the modified enzymes suggested steric hindrance between
Our previous paper13) showed that the interaction of SepII with a2M occurred in a
the modified enzymes and the insoluble pro-
immunogenicand maybe of value for the treatment of venous blood clots.
molar ratio of2 : 1. A stoichiometry of close to 2: 1 was reported for the reaction between trypsin or chymotrypsin and a2M by Bjork et al.29) Steiner et al. reported that the equimolar a2M-thrombincomplexcontains a second site that is not readily cleaved by thrombin but is accessible to trypsin. Their results showed that the a2M-thrombincomplexwasno longer a
modified
good substrate for thrombin but was a good substrate for trypsin. Steiner et al.30) assumed
tein
substrate,
fibrin,
the
highly
modified
PEG2-SepII-L had a similar level of activity to that of the less modified PEG2-SepII-S. highly solves
Fifty
modified PEG2-SepII-L, fibrin, is almost certain
percent
loss of the activities
PEG2-SepII-S
and
The
which disto be non-
of the
PEG2-SepII-L
complexes with the protein proteinase inhibitor, SSI, are shown in Fig. 6. The PEG2-
SepII-S and PEG2-SepII-L preparations
show-
ed higher resistance to the SSI inhibition than that of the native one. The slow interaction of PEG2-SepII-S and PEG2-SepII-L might be caused by steric hindrance between the PEG2-modified enzymes and proteinaceous inhibitor SSI. At pH ll.5 and 30°C, inactivation of the PEG2-SepII-S and PEG2-SepII-L was observ-
that this could result from a conformational alteration occurring at the active site as a consequence of the conformation of thrombin or could simply be the result of steric hindrance. Miyata et al. reported an equimolar ratio for Serratia sp. protease with a2M.31) This study showed that the interaction of PEG2-SepII-S with a2M occurred in a molar ratio
of 1 : 1, although
the
modified
PEG2-
SepII-S and SepII-L showed similar substrate specificity towards small fluorogenic and chroed with the first order rate constants, k, of mogenic substrates as shown in Table I. The 0.0023 sec"1 and 0.0013 sec"1, respectively. differences of the two, the modified enzyme These values demonstrated that there is an and native one, were molecular weights, the effect on the modification of SepII with PEG2, former was 170,000 and the latter was about and that the PEG2-SepII-L was more stable in 23,000. The previous results13) from the differalkaline pH around ll.5 than the free SepII. ence spectra at 294nm showed that conforThe stability of the PEG2SepII-S and PEG2- mational change in the a2Mmolecule due to SepII-L might be caused by a decrease of self- the binding to SepII is completed within 30 sec, digestion of the modified enzyme molecules. It while this paper showeda positive and obtuse would be useful to increase the alkaline stabilpeak at or within 1 min, but no peaks observed ity of SepII with activated PEG2 molecules. after 3 min with PEG2-SepII-S versus a2M. We propose from these results that a molar ratio Interaction of (X2Mwith high molecular weight of the interaction of proteolytic enzymes with PEG2 - SepII- S a2Mmay depend on the molecular weight and Proteolytic activity seems to be necessary for surface condition of the proteolytic enzymes. entrapment inside
a2M and the entrapment
mechanismwas explained by the "trap hypothesis" proposed by Barrett and Starky.28) According to the "trap hypothesis," binding is
initiated by limited proteolytic attack on the "bate region," near the middle of the four
identical
formational
a2Msubunits, which results in conchanges such that
molecule is irreversibly molecule.
the enzyme
trapped within the a2M
Acknowledgments.
This
research
was supported
in
part by a Grant-in-Aid for Scientific Research from the Ministry of Agriculture, Forestry, and Fisheries of Japan as an original
and creative
project
in biotechnology.
We
wish to thank Professor S. Murao, Department of Applied
Microbial Technology, the Kumamoto Institute of Technology, and Associate Professor K. Oda, Department of Agricultural Chemistry, University of Osaka Prefecture, for the kind gift of Streptomyces subtilisin inhibitor (SSI). We thank Mr. T. Sato for the operation of the electron microscope.
2071
Functional Changes of PEG-modified Serine Proteinase from Aspergillus R. Fields, Biochem. J., 124, 581 (1971). M. Roth, Anal. Chem., 43, 880 (1971).
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