Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction.

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.     

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the heparin-antithrombin III/thrombin reaction by active site blocked-thrombin.

Active site blocked-thrombin, prepared by reacting thrombin with valyl-isoleucyl-prolyl-arginine chloromethyl ketone, inhibits the heparin enhanced-antithrombin III/thrombin reaction. Since active site blocked-thrombin does not interact with antithrombin III it was concluded that active site blocked-thrombin was competing for heparin in the reaction system. The heparin concentration dependence for maximum enhancement of the antithrombin III/thrombin reaction in the presence and absence of active site blocked-thrombin indicated that heparin was binding to thrombin to enhance the reaction rate. A dissociation constant value of 6.4×10^?9M was estimated for the heparin·thrombin complex which is similar to the value of 5.8×10^?9M previously reported (Griffith M.J. (1979)$\text{J. Biol. Chem.}$ in press). Antithrombin III·thrombin complexes were also found to bind heparin with an affinity equivalent to thrombin. The results were interpreted to indicate that heparin binds to thrombin as the first step in the mechanism of action of heparin in enhancing the antithrombin III/thrombin reaction.     

The site of linkage of a retinoid affinity label to plasma retinol-binding protein

The retinoid affinity label 11[³H]--ionylidene ethylbromoacetate (IEBA) was covalently bound to plasma retinol-binding protein (RBP) and studies were conducted to identify the region of the protein molecule that contained the linkage between the IEBA ligand and RBP. Cleavage by trypsin and cyanogen bromide of the labeled protein followed by high-performance liquid chromatography (HPLC) separation of peptides and identification of radioactive peaks by amino acid analysis points to attachment of the ligand on tryptic peptides T(1+2) (containing residues 1–5) and T(21) (residues 156–163). These two peptides in the native protein molecule are connected by a disulfide bond between Cys-4 and Cys-160. To confirm the site of attachment of the radioactive ligand, unreduced IEBA-RBP with the disulfide bonds intact was treated first with cyanogen bromide and then with trypsin. Separation of the tryptic peptides by HPLC yielded one main peak of radioactivity containing both peptides T(1+2) and T(21), presumably connected by a disulfide bond. Taken together, these results indicated that the sites of attachment of IEBA to RBP are located within the region of the RBP molecule close to the Cys-4–Cys-160 bond, and specifically within the region comprised of amino acid residues 1–5 and 156–163.     

Formation and stability of the complex formed between human antithrombin-III and thrombin

The inhibition of thrombin by antithrombin-III involves formation of a 1:1 covalent complex between protease and inhibitor and concomitant cleavage of the antithrombin-III peptide chain after Arg-385. The resultant fragment remains connected to the complex via a disulfide bond. This complex spontaneously breaks down into a fragment of approximately 55,000 daltons and smaller peptides. Breakdown is prevented by the presence of hydroxylamine or diisopropylflurophosphate, or by denaturation with urea. It occurs even if the purified complex is treated with diisopropylflurophosphate prior to purification, and can be greatly accelerated by the presence of small amounts of active thrombin. The initial sites of proteolytic attack on the complex are after Arg-13 of the thrombin A chain and Arg-68 of the thrombin B chain. These data indicate that active thrombin can be released from the antithrombin-thrombin complex, and that thrombin becomes more susceptible to proteolytic attack when complexed with antithrombin.     

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding affinity of heparin for thrombin

The present study has shown that calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction. The initial rate of thrombin (4.0 nM) inhibition by antithrombin III (200 nM) in the presence of heparin (2.5 ng/ml) decreased from 3.6 nM/min (in the absence of calcium) to 0.12 nM/min in the presence of 10 mM calcium. In the absence of heparin, the initial rate of thrombin inhibition by antithrombin III was not affected by calcium. The heparin-catalyzed antithrombin III/thrombin reaction is described by the general rate equation for a random-order, bireactant, enzyme-catalyzed reaction (M. J. Griffith (1982) J. Biol. Chem. 257, 13899-13902). As such, the reaction is saturable with respect to both thrombin and antithrombin III. The apparent kinetic parameters for the heparin-catalyzed antithrombin III/thrombin reaction were determined in the presence and absence of calcium. The apparent heparin/antithrombin III dissociation constant values were not measurably different in the presence of 0, 1.0, and 3.0 mM calcium. The apparent heparin/thrombin dissociation constant value increased from 7.0 nM, in the absence of calcium, to 10 and 30 nM in the presence of 1.0 and 3.0 mM calcium, respectively. The maximum reaction velocity, at saturation with respect to both proteins, was not affected by calcium. It is concluded that calcium binds to functional groups within the heparin molecule which are required for thrombin binding.     

Independent responses of nucleoside triphosphatase and protein kinase activities in nuclear envelope following thioacetamide treatment

We have monitored the binding of heparin to thrombin as well as antithrombin by fluorescence techniques. The interaction of mucopolysaccharide with thrombin is characterized by a stoichiometry of 2:1 with K_DISS^H1T = K_DISS^H2T = 8 × 10^?7 M. The interaction of heparin with antithrombin is typified by a stoichiometry of 1:1 with K_DISS^H-A = 5.7 × 10^?8 M. A plot of the initial velocity of the thrombin-antithrombin reaction versus mucopolysaccharide concentration exhibits an ascending limb, descending limb and final plateau. The ascending limb of the kinetic profile corresponds to the level of heparin-antithrombin complex. The descending limb of the initial velocity plot coincides with the formation of tertiary complexes between two molecules of heparin and one molecule of thrombin. Thus, our data demonstrates that the generation of heparin-antithrombin complex is responsible for the mucopolysaccharide dependent acceleration of enzyme neutralization. Furthermore, our results also suggest that interactions between heparin bound to inhibitor and free enzyme can account for only a small fraction of the total kinetic effect of the complex carbohydrate. Therefore, we conclude that the direct binding of heparin to antithrombin is probably responsible for the anticoagulant effect of the mucopolysaccharide.     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction and thermal stability of fluorescent labeled derivatives of thrombin and antithrombin III

Derivatives of human thrombin and antithrombin III with fluorescent labels covalently attached to their carbohydrate moieties were prepared by reaction of periodate-oxidized proteins with amino derivatives of dansyl, fluorescein and pyrene. The labeled derivatives retained full biological activity, including their ability to form stable enzyme-inhibitor complexes, a reaction whose rate could be monitored by the increase in fluorescence polarization. When the dansyl-labeled derivatives were heated, they exhibited sigmoidal increases in polarization with midpoints near 50 degrees C for thrombin and 60 degrees C for antithrombin III. By contrast, a complex between antithrombin III and dansyl-thrombin showed no change in polarization up to 70 degrees C, suggesting that the individual components are more stable in the complex. These studies show that fluorescent labels attached to carbohydrate moieties of glycoproteins provide convenient probes for monitoring conformational changes and protein-protein interactions with minimum interference by the probe.     

Ca2+-induced structural change in the Ca2+Mg2+ domain of troponin C detected by crosslinking

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca²⁺. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca²⁺, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca²⁺, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the α-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the α-carbon distance His-125-Lys-136, do not exceed 14 Å. Comparison with the X-ray structure of troponin C (Herzberg, O, and James, M.N.G. (1985) Nature 313, 653–659) indicates that some of the above distances increase on Ca²⁺-binding.     

A fragment of antithrombin that binds both heparin and thrombin.

In order to identify the regions of antithrombin that interact with heparin and thrombin, it was degraded with CNBr and the activities of the isolated products were investigated. These fragments did not exhibit direct thrombin-neutralizing activity; however, one unique fragment was found to bind to heparin-Sepharose and also to interfere with the inhibition of thrombin by intact antithrombin. This fragment was identified as the one consisting of three disulphide-linked polypeptide chains containing residues 1-17, 104-251 and 424-432. At a concentration of 46 nM, this product decreased the heparin-enhanced thrombin-inhibitory activity of antithrombin by half, and completely abolished this inhibition when above 300 nM. In the absence of heparin, the action of antithrombin was not completely nullified by the fragment, even when present at relatively high concentrations. At a given fragment concentration, the extent of inhibition was independent of antithrombin concentration over the range tested. It was found that the fragment decreased the second-order rate constant for the antithrombin-thrombin reaction. Reduction and alkylation of the fragment showed that the above properties reside primarily in the peptide with residues 104-251. It is concluded that this peptide possesses portions of the antithrombin molecule that bind to heparin as well as to a site on thrombin.     

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor

A bicyclic hexadecapeptide, which corresponds to the sequence 36-51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-chymotryptic activity with a Ki of 7.1.10(-7) M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a Ki of 4.3.10(-5) M, indicating the broad specificity of this ring system.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.     

Comparison of the kinetic behavior of human and bovine proteins in the heparin-catalyzed antithrombin III/thrombin reaction

Significant differences between saturation kinetic properties of heparin-stimulated reactions between thrombin and antithrombin III from human and bovine species were observed. In both systems, the apparent Km for antithrombin III was higher than the KD for antithrombin III-heparin interaction, monitored by intrinsic protein fluorescence change. The Km for thrombin and kcat were much higher for proteins of the human species than the bovine species. The apparent Km for one human protein was dependent on the concentration of the other human protein, indicating interaction of the binding events. The reaction product formed from the bovine proteins was a potent inhibitor of the reaction but the product from the human proteins was a poor inhibitor. The major differences between the two species appeared to be related to interaction of thrombin or thrombin derivatives with heparin or heparin-antithrombin III complexes.