Comparison of a recombinant trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos

The objective was to compare the effects of treating bovine semen with two trypsin products (the porcine pancreas extract and a recombinant) and a control (no trypsin) on in vitro embryo production. Our hypothesis was that the trypsin treatments would not cause any significant difference in fertilization and embryo development as compared to the control. Semen was washed through a gradient system containing a porcine-origin trypsin, a recombinant bovine-sequence trypsin, or the control (no trypsin). Oocytes (n=3036) were collected from abbatoir-derived ovaries, matured for 24h, and allocated into three groups: porcine trypsin (n=1040), recombinant trypsin (n=972), and control (n=1024). Ova were inseminated with 1 x 10(6) motile sperm/mL and cultured for 18-24h. Thereafter, presumptive zygotes were cultured for 7 days in 50 microL G1/G2 micro-droplets under mineral oil. Overall, sperm motility was lower before than after each treatment (mean of 51.4% versus 70.2%, respectively; P<0.001); however, motility was not significantly different among the three groups (porcine-origin trypsin=68.8%, recombinant trypsin=69.0%, and control=72.6%). Similarly, there was no significant difference among these groups for cleavage rates (70.1, 70.9, and 73.9%), or the number of morula/blastocyst stage embryos (53.4, 53.3, and 48.7%). In conclusion, treatment of bovine sperm with either porcine-origin trypsin or recombinant trypsin prior to insemination had no detrimental effects on in vitro embryo development.     

Sodium periodate stimulation of normal and chronic lymphocytic leukemia lymphocytes : II. The use of neuraminidase and trypsin to probe the nature of the response

Sodium periodate stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. The role exerted by cell surface components in this response was investigated by pre-treatment or post-treatment of cells with either neuraminidase or trypsin. Both enzymes substantially reduced the sodium periodate response of normal and leukemic lymphocytes when they were added after the mitogen. In normal lymphocytes when the treatment sequence was reversed (enzyme before sodium periodate), neuraminidase had no effect while trypsin greatly potentiated the action of this mitogen. In leukemic lymphocytes, however, neuraminidase reduced the response, whereas trypsin had little or no effect. In comparison with another mitogen, the response of normal and leukemic lymphocytes to phytohemagglutinin was not significantly altered by pre-treatment with neuraminidase or trypsin. Collectively, these results imply that significant differences exist in the cell surface of these two types of lymphocytes and that the oxidation of cell surface components other than sialic acid are involved in the response of these cells to sodium periodate.     

Factors involved in the intestinal feedback regulation of pancreatic enzyme secretion in the rat.

Further studies on the feedback regulation of pancreatic enzyme secretion by trypsin were conducted in conscious rats, surgically prepared so that pancreatic juice could be collected or returned. Suppression of enzyme secretion by trypsin as well as its stimulation by SBTI occurred only in the upper part of the small intestine, where the hormone CCK is known to be released. Over a limited range, trypsin suppression of pancreatic secretion was proportional to the dose of trypsin. Higher concentrations had no further effect, suggesting "saturation" of the intestine. Trypsin which had its active center blocked by DFP did not suppress enzyme output. These results supported the concept that only trypsin (or chymotrypsin) with an exposed active center suppressed pancreatic enzyme secretion in the rat by somehow suppressing the release of CCK from the intestinal cell. Presumably CCK is released from the intestine following "removal" of trypsin from the intestine either by diverting the juice or by feeding SBTI which binds the enzyme. All of the evidence supported the view that the effect of trypsin or SBTI on pancreatic secretion was mediated at the intestinal level and not in the blood as has been suggested.     

Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases.

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16--100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.     

用定位突变探讨胰蛋白酶的稳定性

应用定位突变的方法,对鼠胰蛋白酶分子中易自溶位点的氨基酸残基进行改造,以探索提高胰蛋白酶稳定性的可能。将鼠胰蛋白酶原cDNA从质粒pTRAP上切下,插入载体M13mp8,在E.coli JM 101菌株中转化、复制后用以转染RZ1032缺陷型菌株,利用含U模板,进行按实验目的设计寡聚核苷酸引物引导的定位突变,将易自溶位点Ary105改造为Leu或Gly。经酶切和序列测定,证明在设计位点发生了预期的碱基突变。为了检查活性方便,又将含突变型胰蛋白酶cDNA从pTRAP上切下,插入另一个表达载体pTN中,转化入E.coli SM 138宿主中进行表达,表达产物分泌到围膜间隙,作专一性底物TAME活性胶方法检测,证明改造后的表达产物具有胰蛋白酶活性。对突变与野生型胰蛋白酶进行了初步比较。     

Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme

In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.     

Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin.

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.     

Protease-Induced Formation of the Sperm Acrosomal Filament

Filament extension during the sperm acrosome reaction in Sicyonia ingentis is triggered by an egg trypsin-like protease whose action can be mimicked using trypsin. Using biotinylated trypsin and either a fluorescently-labeled or colloidal gold-labeled antibody to biotin, trypsin binding was localized to the anterior granule of the sperm which is exposed upon acrosomal exocytosis. The binding was to proteinaceous material at the base of the granule juxtaposed to the inner acrosomal membrane. Other labeled proteins also bound in the same pattern but only in the presence of unlabeled trypsin; non-proteolytic proteins did not induce filament formation. Binding of all proteins tested occurred slowly over a period of about 30 min. A minimum of 30 min of trypsin exposure was required in order to trigger filament formation, and increasing trypsin concentration did not reduce this time requirement. These results indicate that the protease slowly uncovers a binding site for itself (or other proteins), and then its proteolytic activity is again required to induce filament formation. The protease kallikrein appeared to be a more potent inducer than trypsin, while thrombin and clostripain had no apparent inducing activity.     

Studies on the influence of Trasylol on the partition of trypsin between the human plasma protease inhibitors in vitro.

The distribution of trypsin between the protease inhibitors of human serum with and without Trasylol was studied in vitro. 1) Trypsin was preferentially bound by alpha2-macroglobulin on addition of small amounts of the enzyme to normal serum in both the presence and absence of Trasylol in a molar concentration equal to that of alpha2-macroglobulin. 2) On saturation of alpha2-macroglobulin, a considerable amount of trypsin was bound by Trasylol even when most of the serum alpha1-antitrypsin was in a free form. 3) In reaction mixtures containing small amounts of trypsin, Trasylol was identified in a free form as well as in complex with trypsin-alpha2-macroglobulin complex and to a limited extent with trypsin. 4) With larger amounts of trypsin, sufficient to saturate alpha2-macroglobulin, increasing amounts of Trasylol were bound to trypsin. The relative amount of Trasylol bound to trypsin-alpha2-macroglobulin complexes was now smaller. This was explained by a higher affinity (or binding rate) of Trasylol for trypsin than for trypsin-alpha2-macroglobulin complexes. 5) Trypsin-Trasylol complexes showed no signs of dissociation after 5 h incubation at 37 degrees C in serum.     

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.     

Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae,Spodoptera frugiperda

A dose‐dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl‐L‐lysine chloromethyl ketone‐HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane‐bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross‐class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross‐class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available. © 2010 Wiley Periodicals, Inc.     

Modification of the trypsin-dependent cleavage activation site of the human metapneumovirus fusion protein to be trypsin independent does not increase replication or spread in rodents or nonhuman primates

The contribution of cleavage activation of the fusion F protein of human metapneumovirus (HMPV) to replication and pathogenicity in rodents and nonhuman primates was investigated. Recombinant HMPVs were generated in which the naturally occurring trypsin-dependent cleavage sequence (R-Q-S-R downward arrow) was replaced by each of three sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multibasic sequences derived from avian metapneumovirus type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R downward arrow). The third one (R-Q-P-R) was derived from a recently described trypsin independent HMPV isolate (J. H. Schickli, J. Kaur, N. Ulbrandt, R. R. Spaete, and R. S. Tang, J. Virol. 79:10678-10689, 2005). To preclude the possibility of conferring even greater virulence to this significant human pathogen, the modifications were done in an HMPV variant that was attenuated by the deletion of two of the three envelope glycoproteins, SH and G. Each of the introduced cleavage sequences conferred trypsin independent F cleavage and growth to HMPV in vitro. However, they differed in the efficiency of trypsin independent growth and plaque formation in vitro: R-R-R-R > R-K-A-R > R-Q-P-R > R-Q-S-R. The R-R-R-R mutant was the only one whose growth in vitro was not augmented by added trypsin, indicative of highly efficient trypsin independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract between the mutants, and virus was not detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence.     

Facilitation of electrofusion of mouse lymphoma cells by the proteolytic action of proteases

Cell fusion of mouse lymphoma (L5178Y) was achieved by applying electrical pulses under dielectrophoresis. The presence of dispase, pronase or trypsin facilitated the electric pulse-induced cell fusion. Heat-inactivated pronase was no longer effective. Protease inhibitors (aprotinin and p-tosyl-L-lysine chloromethylketone) suppressed the effect of trypsin. Even in the absence of proteases, the cells pretreated with dispase or pronase underwent fusion with high probabilities, as far as free calcium ions were present in the external solution. It is concluded that facilitation of electrofusion by proteases is due to their proteolytic activities.     

Trypsin cleavage of the alpha-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable)nucleotide binding in beef heart mitochondrial F1-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103-7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the alpha-subunit of F1 is altered by the mild trypsin treatment, whereas no change was detected in beta-, gamma-, delta- or epsilon-subunits. Binding of ADP to the trypsin-treated F1 was compared to binding to control enzyme over a range of 0-40 muM ADP in a 30 min incubation period. There was no difference between the two enzymes, KADPd in Mg2+ -containing buffer was about 2 muM in each. Since the tight (nonexchangeable)sites are abolished in trypsin-treated F1, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the alpha-subunit on beta-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the alpha-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F1 but leaves partial functions intact.     

ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four residues located within the core "alpha-crystallin" domain, Lys(82), Lys(103), Arg(116), and Arg(123), that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), located near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K(m) for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.     

Effect of Trypsin on the Vernalization Process in Winter Wheat

This paper deals with the experimental results of the effect of trypsin on the vernalization process in the winter wheat. The variations of both the trypsin-like enzyme activity and the soluble protein content during vernalization in winter wheat seedlings were assayed. The results are as follows: 1. When the vernalization was progressing to the middle stage (around 25 days), the seedlings of the winter wheat were moved into the room temperature for continuous culture. These seedlings possessed the ability to earing after this kind of treatment, but earing development was rather late. Whereas the development of the earing was much earlier by treating them with 100 ppm trypsin just after moving into the room temperature condition. 2. Earing could not be induced by treatment with trypsin (100 ppm) in nonvernalized winter wheat. 3. Vernalization process was promoted in the initial period by trypsin under the low temperature, but it was inhibited in the middle and there was no remarkably effect on the developmental process in the later period. 4. There was no effect by the treatment of trypsin on the spring wheat under the conditions with or without vernalization. 5. The trypsinlike enzyme activity during the cold codication was increased in the initial period, then decreased remarkably later, but at the same time the soluble protein content increased rapidly. These results indicate that at the middle-later period of the vernalization the synthesis of some specific proteins is very important for the proceeding of varnalization.     

The reversible thiol-disulphide exchange of trypsin and chymotrypsinogen with a tumour-derived inhibitor. Kinetic data obtained with fluorescein-labelled polymeric collagen fibrils and casein as substrates.

Ehrlich ascites cells contain a cytoplasmic inhibitor of both trypsin and the granule neutral protease and possess a reactive thiol which interacts with an important disulphide bond in trypsin, resulting in the formation of the trypsin-inhibitor complex. When a fixed quantity of trypsin was completely inhibited by addition of the cytoplasmic inhibitor, the trypsin could be re-activated by the addition of either trasylol-trypsin or chymotrypsinogen. Since trasyloltrypsin, chymotrypsinogen (and any derived chymotrypsin) has no ability to solubilise fluorescein-labelled peptides from the substrate, the appearance of trypsin activity was probably due to a non-enzymic exchange reaction, in which these inactive forms displaced trypsin from the trypsin-inhibitor complex. Kinetic data suggest that this displacement was a time-dependent equilibrium reaction controlled by the relative concentration of the reacting species.     

胭脂鱼胰蛋白酶cDNA 克隆以及不同蛋白含量日粮和饥饿对mRNA 表达和酶活力的影响

为检测不同蛋白含量的日粮和饥饿对胭脂鱼(Myxocyprinus asiaticus)幼鱼胰蛋白酶活性和mRNA表达的影响, 首先用RACE 和PCR 的方法从胭脂鱼幼鱼的肝胰脏组织中克隆得到胰蛋白酶cDNA 全长, 然后用半定量RT-PCR 和酶活性检测方法分别检测了经不同蛋白含量日粮(酪蛋白含量分别为35%、45% 和 55%)投喂和饥饿处理后的胭脂鱼幼鱼的胰蛋白酶mRNA 表达水平和胰蛋白酶活力的变化。结果显示, 胭脂鱼胰蛋白酶cDNA 全长为912 bp。投喂蛋白质含量适中(45%酪蛋白)日粮组的试验鱼胰蛋白酶活性和mRNA 水平高于投喂高蛋白水平日粮组(55%酪蛋白)和低蛋白水平日粮组(35% 酪蛋白); 饥饿明显降低mRNA水平和胰蛋白酶活性; 胰蛋白酶活性的变化滞后于mRNA 水平的变化。胰蛋白酶活力的变化与mRNA 水平的变化之间未呈现直接相关性。因此, 胭脂鱼胰蛋白酶合成可能是一个由多种因素共同调控的复杂过程。       

MOLECULAR CLONING OF TRYPSIN AND THE EFFECT OFDIETARY PROTEIN LEVELS AND STARVATION ON TRYPSINmRNA EXPRESSION AND ENZYME ACTIVITY IN JUVENILECHINESE SUCKER (MYXOCYPRINUS ASIATICUS BLEEKER)

The effect of dietary protein and starvation on the expression of trypsin was evaluated in the Chinesesucker (Myxocyprinus asiaticus Bleeker). The complete trypsin cDNA was cloned from juvenile Chinese suckerpancreatic tissue by using RACE and PCR methods. We used semi-quantitative RT-PCR and enzymatic activitymeasurements to quantify mRNA expression and trypsin activity in fish that were either starved or fed differinglevels of dietary casein (35%, 45% and 55%). The results showed that the Chinese sucker trypsin cDNA sequencewas 912 bp in length. Trypsin activity and mRNA levels were higher in fish that were fed moderate (45% casein)levels of protein than those that were fed high or low levels. Starvation significantly decreased mRNA expressionlevel and trypsin activity. The changes in trypsin activity tended to lag behind the changes in mRNA levels. Therewas no direct relationship between the trypsin activity and mRNA level. Given this, the trypsin synthesis is acomplex process regulated by a balance of several factors in the Chinese sucker.     

Investigation of the interaction of trypsin with heparin

The reversible inhibition of the enzymatic activity of trypsin by heparin was investigated. On the basis of an analysis of the Lineweaver-Burk and Dixon graphs, a noncompetitive nature of the inhibition of the BAPA amidase activity of trypsin by heparin was detected, and the values of Km and Ki were determined, equal to 3.1 . 10(-4) and 3.7-3.9 . 10(-7) M, respectively. A comparison of these values indicates a great affinity of heparin for the enzyme. It was shown that heparin inhibits the BAEE esterase activity of trypsin and at the same time has no inhibiting effect on acetyltrypsin. Considering that the acetylation of trypsin leads to selective blocking of the epsilon-amino groups, it was concluded that the epsilon-amino groups of the lysine residues of the trypsin molecule participate in the interaction with heparin.