Bovine Plasmin Research Articles

A rapid and sensitive biosensor for measuring plasmin activity in milk

Plasmin play an important role in milk spoilage and can cause quality issues in ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring plasmin activity in milk have low sensitivity and are too slow to be used in routine testing. We report a bioluminescence resonance energy transfer (BRET)-based biosensor that can measure naturally occurring low levels of plasmin activity in milk within a few minutes. The biosensor incorporates a plasmin-specific peptide target sequence flanked by the resonance energy BRET transduction elements: green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the peptide linker sequence by human plasmin led to an approximately 87% decrease in the starting BRET ratio. Using a 10 min incubation time, the detection limit for human plasmin was 0.25 nM and 0.86 nM for bovine plasmin in 50% (v/v) full fat milk with EC50's of 5.89 ± 0.12 nM and 5.97 ± 0.59 nM, respectively. These detection limits are below the naturally occurring levels of plasmin reported to occur in raw or processed cow’s milk. The plasmin biosensor therefore has the potential to measure naturally occurring plasmin levels directly in raw and UHT-processed milk samples. The protease biosensor described herein is selective and sensitive and retains its function in the complex colloidal matrix of milk. It therefore meets many of the requirements for routine use in production settings. We propose its use for managing plasmin-related quality issues in UHT milk and other dairy products.

Effects of stocking density on oxidative stress status and mammary gland permeability in early lactating dairy cows.

The current study was conducted to investigate the effect of stocking density (SD) on oxidative stress status and mammary gland permeability in early lactating dairy cows. Thirty-two dairy cows were allocated into 16 blocks, basing on parity, previous milk yield, and body weight, and were then randomly assigned into one of the two treatments as follows: 75% (75SD) and 100% (100SD) SD. The cows were fed with same diet throughout the 8-week experimental period. The milk yield and milk sample were collected on two consecutive days during the 8-week experimental period weekly. Plasma samples were collected on fourth and eighth experimental weeks. Raw, energy-corrected, and 4% fat-corrected milk yield were significantly higher in 75SD-cows than that of 100SD-animals, respectively. The milk somatic cell count was lower in 75SD-cows than that of 100SD-animals. The levels of Na+ , Na+ /K+ , bovine serum albumin and plasmin were lower in 75SD-cows than those of 100SD-cattle, respectively. The 75SD-cows had reduced insulin and insulin/glucagon levels but higher prolactin and growth hormone concentrations, compared with those of 100SD-animals, respectively. In conclusion, compared with low SD-animals, early lactating cows with higher SD had higher oxidative stress status, which further led to a greater mammary gland permeability.

Streptococcus uberis acquires plasmin activity following growth in the presence of bovine plasminogen through the action of its specific plasminogen activator.

Three (0140J, C197C and EF20) out of four strains of Streptococcus uberis exhibited high levels of bound plasmin activity following growth in the presence of bovine plasminogen. The remaining strain (C197) bound considerably less plasmin following growth in the same medium. In contrast to the others, this strain was unable to activate bovine plasminogen. Following growth of strain C197 in the presence of bovine plasminogen and a source of plasminogen activator (urokinase or culture filtrate from strain 0140J) high levels of bacterially associated plasmin were detected. None of the strains was able to activate human plasminogen and only trace levels of plasmin activity were detected in association with the S. uberis following growth in the presence of human plasminogen. All strains were able to bind plasmin activity following incubation in the presence of either bovine or human plasmin. However, in each case the level of activity detected following incubation in human plasmin was approximately five-fold less than that observed following incubation with bovine plasmin. None of the strains bound detectable levels of either human or bovine plasminogen. It is concluded that activation of plasminogen is required prior to binding of plasmin by S. uberis.

Kinetics of changes in plasmin activity and proteolysis on heating milk

Heat-induced inactivation of bovine plasmin, denaturation of beta-lactoglobulin (beta-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of beta-casein was studied in a model system consisting of phosphocasein and beta-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of beta-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of beta-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65-80 degrees C was its interaction with beta-lg (kr at 60 degrees C, 0.0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin approximately 45% of the beta-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of beta-lg with the casein micelles at 60 degrees C had a rate constant of 3.71 x 10-5 min-1 and an Ea of 259 KJ/mol; thermal denaturation of beta-lg was of much less importance, with a rate constant at 60 degrees C of the order of 1 x 10-10 min-1 and an Ea of 250 KJ/mol. On denaturation of all beta-lg in the system, a maximum of approximately 55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of beta-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of beta-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with beta-lg.

Comparative Study of Enzyme Activity and Stability of Bovine and Human Plasmins in Electrophoretic Reagents, β-mercaptoethanol, DTT, SDS, Triton X-100, and Urea

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Use of the surface proteins GapC and Mig of Streptococcus dysgalactiae as potential protective antigens against bovine mastitis.

Streptococcus dysgalactiae is a significant pathogen associated with bovine mastitis in lactating and nonlactating dairy cows, causing a severe inflammatory response of the mammary gland, which results in major economic losses to the dairy industry. Two proteins from S. dysgalactiae strain SDG8 were tested for their protective capacity against a homologous bacterial challenge in a dry cow model. The first was a bovine plasmin receptor protein (GapC), which shares 99.4% sequence identity to the plasmin-binding Plr protein of group A streptococci. The second protein product was Mig, a alpha2-M-, IgG-, and IgA-binding protein present on the cell surface of SDG8. We investigated the efficacy of immunization with purified recombinant forms of GapC and Mig by measuring the number of somatic cells and assessing the presence of the challenge strain in mammary secretions following challenge. In this model, we found that, although the number of quarters containing SDG8 was significantly reduced in the GapC- but not in the Mig-immunized animals, the somatic cell counts from teat secretions were significantly decreased in both the GapC and Mig vaccinates.

Bovine Protein C Inhibitor Has a Unique Reactive Site and Can Transiently Inhibit Plasmin

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg-Ser) of other species' PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system.

Hydrolysis of porcine β-casein by bovine plasmin and bovine chymosin

The action of bovine chymosin and bovine plasmin on porcine β-casein was studied and compared with their effect on bovine β-casein in an attempt to elucidate the similarities in specificity of these enzymes on porcine and bovine β-caseins. Bovine plasmin rapidly hydrolysed porcine β-casein at Lys106-Arg107, Lys49-Ile50 and Lys168-Val169 to yield γ-caseins. Several peptides that had the same N-terminal amino acid sequence as porcine β-casein were also produced. Plasmin hydrolysis sites determined by separating the pH 4.6-soluble peptides formed on hydrolysis were Lys33-Leu34, Lys49-Ile50, Lys95-Asp96, Lys98-Ala99, Lys106-Arg107, Lys108-Gly109 and Lys168-Val169. Porcine β-casein, like bovine β-casein, was hydrolysed by bovine chymosin into three distinct electrophoretic bands designated porcine β-I-, β-II- and β-III-casein, all of which had the N-terminal sequence of porcine β-casein. Two initial pH 4.6-soluble peptides formed on hydrolysis of porcine β-casein by chymosin had N-terminal amino acid sequences commencing at porcine β-casein amino acid 197, suggesting that porcine β-I-casein corresponds to fragment 1–196, while the sequences of two other small peptides commenced at amino acids 1 and 207. The C-terminal cleavage site(s) leading to formation of porcine β-II and β-III casein were not determined.

The individual or combined action of chymosin and plasmin on sodium caseinate or β-casein in solution: effect of NaCl and pH

Although ~-casein appeared to be degraded by bovine plasmin slightly faster in a salt- free system than in a solution containing NaCI, increasing the concentration of NaCl had little effect on the activity or specificity of the enzyme. The activity, but not the specificity, of bovine plasmin on ~-casein was influenced by pH; the rate of hydrolysis proceeded in the order pH 8.4 > 6.5 > 5.4. Chymosin did not appear to degrade peptides produced from ~-casein by plasmin (i.e. y!' Y2' Y3-caseins and their corresponding proteose peptones). In sodium caseinate, as1-casein was more susceptible to hydrolysis by chymosin than ~-casein. The activityof chymosin on Na-caseinate increased with decreasing pH in the range of 6.5 to 5.4. Increasing concentrations of NaCl inhibited proteolysis to an extent that was dependent on the reaction pH. Bovine plasmin rapidly hydrolysed N a-caseinate to a wide range of peptides detectable by polyacrylamide gel electrophoresis (PAGE) and it was also capable of degrading Na-caseinate-derived peptides produced by chymosin to a sim- ilar range of peptides. © Inra/Elsevier, Paris. chymosin / plasmin / sodium caseinate / ~-casein / hydrolysis

Changes in the bovine zona pellucida induced by plasmin or embryonic plasminogen activator

Effects of bovine plasmin and plasminogen activator recovered from bovine embryo-conditioned medium (bePA) on the polypeptide profile and solubility of bovine zonae pellucidae (ZP) were evaluated. ZP were isolated from bovine ovarian oocytes and incubated at 39°C with 0, 100, or 200 μg/ml plasmin for 0, 24, or 48 hr or bePA with 0 or 100 μg/ml human plasminogen for 0 or 48 hr. ZP were evaluated either by SDS-PAGE or for changes in solubility using a zona pellucida dissolution time (ZPDT) assay. Two prominent polypeptides, molecular weight (MW) 76,000 and 65,000, and two minor polypeptides, MW 23,000 and 22,000, were resolved by SDS-PAGE. No changes occurred in the polypeptide profile for ZP incubated with 0 μg/ml plasmin for 0, 24, or 48 hr, and ZPDT did not differ (P > 0.10). Treatment with 100 or 200 μg/ml plasmin induced reductions in the MW 76,000, 23,000, and 22,000 polypeptides and the appearance of MW 45,000 and <10,000 polypeptides. ZPDT were less (P < 0.05) in 100 and 200 μg/ml compared with 0 μg/ml plasmin. Polypeptide profiles and ZPDT for ZP incubated with bePA were similar (P > 0.10) to ZP incubated with unconditioned medium. Addition of human plasminogen to ZP incubated with bePA reduced the MW 76,000, 23,000, and 22,000 polypeptides, caused the appearance of MW 45,000 and 20,000 polypeptides, and decreased ZPDT (P < 0.05). These results demonstrate that bovine plasmin is capable of proteolytically degrading the bovine ZP and that bePA can indirectly affect the ZP by converting plasminogen to plasmin. Mol. Reprod. Dev. 51:330–338, 1998. © 1998 Wiley-Liss, Inc.

Changes in the bovine zona pellucida induced by plasmin or embryonic plasminogen activator.

Effects of bovine plasmin and plasminogen activator recovered from bovine embryo-conditioned medium (bePA) on the polypeptide profile and solubility of bovine zonae pellucidae (ZP) were evaluated. ZP were isolated from bovine ovarian oocytes and incubated at 39 degrees C with 0, 100, or 200 microg/ml plasmin for 0, 24, or 48 hr or bePA with 0 or 100 microg/ml human plasminogen for 0 or 48 hr. ZP were evaluated either by SDS-PAGE or for changes in solubility using a zona pellucida dissolution time (ZPDT) assay. Two prominent polypeptides, molecular weight (MW) 76,000 and 65,000, and two minor polypeptides, MW 23,000 and 22,000, were resolved by SDS-PAGE. No changes occurred in the polypeptide profile for ZP incubated with 0 microg/ml plasmin for 0, 24, or 48 hr, and ZPDT did not differ (P > 0.10). Treatment with 100 or 200 microg/ml plasmin induced reductions in the MW 76,000, 23,000, and 22,000 polypeptides and the appearance of MW 45,000 and <10,000 polypeptides. ZPDT were less (P < 0.05) in 100 and 200 microg/ml compared with 0 microg/ml plasmin. Polypeptide profiles and ZPDT for ZP incubated with bePA were similar (P > 0.10) to ZP incubated with unconditioned medium. Addition of human plasminogen to ZP incubated with bePA reduced the MW 76,000, 23,000, and 22,000 polypeptides, caused the appearance of MW 45,000 and 20,000 polypeptides, and decreased ZPDT (P < 0.05). These results demonstrate that bovine plasmin is capable of proteolytically degrading the bovine ZP and that bePA can indirectly affect the ZP by converting plasminogen to plasmin.

ELISA for differential quantitation of plasmin and plasminogen in cheese

Bovine plasmin (PLM) and its precursor plasminogen (PLG) are present in cheese, in which the proteinase is thought to play a significant role in proteolysis during ripening. PLM in cheese is usually assayed by enzymic techniques and concentrations are generally expressed as activities. In order to determine absolute concentrations of PLM and PLG in cheese, we have applied an ELISA previously developed for the differential determination of PLM and PLG in milk. The assay used two monoclonal antibodies, one specific for PLG and the other cross reacting with PLM and PLG. PLM concentration was obtained by subtracting the PLG concentration from the value for PLM plus PLG. The assay was evaluated in cheese by determining the total amount of PLG and PLM in milk used in the manufacture of eight semi-hard cheeses on a pilot plant scale, and comparing it with the total amount of PLG and PLM present in the eight corresponding wheys and unripened cheeses. Concentrations of PLG and PLM in 19 commercial cheeses were also determined. It was thus demonstrated that the ELISA made it possible to quantify PLM and PLG in milk and cheese and that PLM and PLG concentrations in cheese were highly dependent on the technology used for cheesemaking.

Characterization of the interaction of bovine plasmin with Streptococcus uberis.

The binding of plasmin to Streptococcus uberis strain 0140 J was optimal in the pH range 5.0-5.5. Plasmin binding decreased exponentially with increasing NaCl concentration (0-0.8 mol l-1), reaching a minimum at NaCl concentrations exceeding 0.55 mol l-1. Neither K+, Mg2+ nor the metal chelator EDTA had any effect on the interaction. Plasmin binding was prevented, in a concentration-dependent manner, by the amino acids lysine, arginine and epsilon-aminocaproic acid. Bound plasmin was also eluted from the bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium in a time- and temperature-dependent fashion, the rate of plasmin loss increased with increasing temperature over the range 4-55 degrees C, and the elution of plasmin from live and heat-killed bacteria was similar. Cell-bound plasmin was only partially inhibited by the physiological inhibitor alpha 2-antiplasmin whereas the serine protease inhibitor aprotinin, and the active site titrant p-nitrophenyl-p-guanidiniobenzoate, inhibited the activity of the cell-bound plasmin by more than 95%.

Effect of Psychrotrophic Microorganisms on the Plasmin System in Milk

The psychrotrophic bacteria that are present in raw milk produce heat-stable proteases that affect the plasmin system and, in turn, the quality of the processed milk and other dairy products. Three bacterial strains, Pseudomonas spp. SRM21A and SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of ∼103 cfu/ml into reconstituted nonfat dry milk and incubated at 4°C for up to 9 d. From each sample, casein and whey fractions were obtained and electrophoresed to visualize protein hydrolysis and plasmin activity. Colorimetric assays were used to quantify plasmin-related activities. The casein SDS-PAGE gels showed that, by 5 d of incubation, caseinolytic activity was visible in whey fractions of all three strains at the same molecular mass range as commercial bovine plasmin, which was used as a control. Colorimetric assays indicated that plasmin activity in the casein samples decreased as incubation time increased; plasmin activity in the whey samples increased, peaking at 5 to 7 d. Plasminogen activity decreased over time in the casein fraction and increased in the whey fraction. Protein immunodetection indicated crossreactivity between anti-bovine plasminogen and the plasmin and plasminogen from whey samples collected at 5 and 7 d from the incubated milk. The growth of the Pseudomonas strains tested and their concomitant production of proteases caused the release of plasmin and plasminogen from the casein micelle into the whey fraction.

The interaction of Streptococcus dysgalactiae with plasmin and plasminogen

The activation of plasminogen and the binding of plasmin by bacteria may have many effects which promote infection. The occurrence of such activities in streptococci is well documented; however, these are yet to be demonstrated for S. dysgalactiae. Consequently, the ability of this bacterium to activate mammalian plasminogen and bind either plasmin or its zymogen was investigated. Activation of bovine plasminogen was dependant on both the strain and the growth medium used for cultivation. Eighteen strains were able to activate bovine and ovine plasminogen and some of these also activated plasminogen from the horse, rabbit and pig. None activated human plasminogen and one strain (CE127) did not activate plasminogen from any source. Tricine-SDS PAGE and zymographic analysis of culture supernatants showed that bovine plasminogen was activated by four out of six strains at two locations corresponding to 16 kDa and 10 kDa. Following the growth of five strains in the presence of bovine plasminogen, all but strain CE127 bound high levels of plasmin activity. In contrast, following growth in human plasminogen none of the strains exhibited bound plasmin activity although all could bind human plasmin directly. All strains were also able to bind bovine and human plasminogen in such a way as to allow its activation by urokinase. We conclude that S. dysgalactiae is capable of activating mammalian plasminogen in a species-specific fashion and that the bacterium is also capable of binding plasmin and plasminogen with an apparent preference for bovine plasmin over human plasmin and/or plasminogen from either species.

Structural Features of Helical Aggregates of Antibacterial Peptides via Simulated Annealing and Molecular Modeling

A 27-residue stretch of amino acids encompassing two putative 13-residue amphiphilic helical segments is an important determinant of activity in the 47-residue antibacterial peptide bovine seminal plasmin. Synthetic peptides corresponding to the 27-residue stretch (P27) SLSRYAKLANRLANPKLLETFLSKWIG as well as the 13-residue segments PKLLET- FLSKWIG (SPF), exhibit antimicrobial activity. An analog of SPF where E has been replaced by K(SPFK) showed improved antimicrobial properties as compared to SPF. The peptides have the ability to bind and permeabilize membranes. We have modeled helical bundles of P27 and the two 13-residue peptides SPF and SPFK using simulated annealing via molecular dynamics. Octameric but not hexameric aggregates of P27 can form channels which would allow the passage of ions. In the case of 13-residue peptides, aggregates formed by 6 monomers can conceivably form ion conducting channels. Since the ability to form channels which would allow the passage of ions across the membranes is an important determinant of the biological activities of these peptides, knowledge of the pore forming structures should help in the design of analogs with improved activities.

Heterologous gene expression of bovine plasmin in Lactococcus lactis.

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active.

Streptococcus uberis acquires plasmin activity following growth in the presence of bovine plasminogen through the action of its specific plasminogen activator

Three (0140J, C197C and EF20) out of four strains of Streptococcus uberis exhibited high levels of bound plasmin activity following growth in the presence of bovine plasminogen. The remaining strain (C197) bound considerably less plasmin following growth in the same medium. In contrast to the others, this strain was unable to activate bovine plasminogen. Following growth of strain C197 in the presence of bovine plasminogen and a source of plasminogen activator (urokinase or culture filtrate from strain 0140J) high levels of bacterially associated plasmin were detected. None of the strains was able to activate human plasminogen and only trace levels of plasmin activity were detected in association with the S. uberis following growth in the presence of human plasminogen. All strains were able to bind plasmin activity following incubation in the presence of either bovine or human plasmin. However, in each case the level of activity detected following incubation in human plasmin was approximately five-fold less than that observed following incubation with bovine plasmin. None of the strains bound detectable levels of either human or bovine plasminogen. It is concluded that activation of plasminogen is required prior to binding of plasmin by S. uberis.

Stabilization of plasmin by lysine derivatives

Plasmin is a serine protease with trypsin-like specificity and is activated from plasminogen by several plasminogen activators. Since plasmin has lysine binding site in its heavy chain, lysine derivatives react with plasmin and then modify its activity. The effects of lysine derivatives such asϵ-aminocaproic acid (EACA) and tranexamic acid on bovine plasmin activity were investigated. In the absence of lysine derivatives, the bovine plasmin activity which was evaluated as the amidolytic activity was reduced in a time- or temperature-dependent manner. However, the bovine plasmin activity became stable upon adding EACA or tranexamic acid. When plasmin was incubated at 4°C for 1, 3 or 5 days without lysine derivatives, the plasmin activity decreased to 43.9%, 19.9% and 11.9% of the initial activity, respectively. On the other hand, when plasmin was incubated at 37°C for 1, 3 or 5 days with tranexamic acid, its activity remained at 110%, 95.6% and 85.9%, respectively. After bovine plasmin had been incubated for 5 days at 4°C in the absence of tranexamic acid, the plasmin activity declined to less than 20%. However, when bovine plasmin had been incubated for 5 days at 37°C in the presence of tranexamic acid, the residual plasmin activity was more than 80%. A similar effect of EACA on bovine plasmin was observed, but it was weaker than that of tranexamic acid. Reversed-phase HPLC followed by SDS-PAGE demonstrated that bovine plasmin was degraded into several fragments. Amino acid sequencing of these fragments revealed that the Lys77-Arg78 or Arg78-1109, Arg342-Met343 and Arg557-Ile558 peptide bonds in the bovine plasmin molecule were cleft, respectively. Only the fragment consisting of the amino acid region from Met343 to the C-terminal amino acid, Asn786, exhibited amidolytic activity. In proportion to inactivation of the bovine plasmin, this fragment disappeared. The above findings suggest that lysine derivatives react with bovine plasmin and then stabilize the activity of plasmin by preventing the degradation of active fragment (Met343-Asn786).

Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin.

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.