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Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.
Bock PE
(1992) J Biol Chem 267: 14974-81
MeSH Terms: Amino Acid Chloromethyl Ketones, Binding Sites, Blood Coagulation Factors, Catalysis, Electrophoresis, Polyacrylamide Gel, Enzyme Precursors, Fluorescent Dyes, Humans, Hydrolysis, Peptide Fragments, Prothrombin, Substrate Specificity, Thrombin
Show Abstract · Added January 20, 2015
The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling of the proteinase active site with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl or N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, followed by selective modification of the NH2OH-generated thiol group on the covalently incorporated inhibitors with each of eight thiol-reactive fluorescence probes. The changes in probe fluorescence intensity of the derivatives, signaling changes in the environment of the catalytic site associated with fragment 2 binding, appeared to be a unique and unpredictable function of the structure of the probe and the connecting peptide. These results demonstrated the utility of the labeling approach for overcoming the problem of not being able to predict which fluorescent label will provide the most useful proteinase derivative for investigating an interaction by enabling a greater variety of them to be prepared and screened for those with the most desirable properties. To determine whether the approach could be extended to other proteinases, the specificity of labeling with the fluorescence probe iodoacetamide, 5-(iodoacetamido)fluorescein, by use of the two thioester inhibitors was evaluated for several other blood coagulation proteinases and related trypsin-like enzymes. All of the proteinases were labeled in an active-site-selective manner. The combined results of quantitating the labeling reactions for the proteinase and inhibitor combinations studied thus far showed active-site-specific incorporation of 0.98 +/- 0.10 mol of inhibitor/mol of active sites and 0.92 +/- 0.11 mol of probe/mol of active sites, representing an overall greater than or equal to 93% site-specificity of labeling. These results demonstrated the broad applicability of the labeling approach for fluorescence studies of proteinases that differ greatly in their catalytic specificities.
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13 MeSH Terms
Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.
Bock PE
(1992) J Biol Chem 267: 14963-73
MeSH Terms: Acetylation, Alkylation, Amino Acid Chloromethyl Ketones, Binding Sites, Electrophoresis, Polyacrylamide Gel, Enzyme Precursors, Fluorescent Dyes, Humans, Hydrolysis, Kinetics, Prothrombin, Substrate Specificity, Thrombin
Show Abstract · Added January 20, 2015
In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.
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13 MeSH Terms
Distribution of prothrombin carbohydrate units upon thrombin activation.
Hudson BG, Heldebrant CM, Mann KG
(1975) Thromb Res 6: 215-21
MeSH Terms: Catalysis, Enzyme Activation, Fucose, Galactosamine, Galactose, Glucosamine, Humans, Mannose, Prothrombin, Sialic Acids, Thrombin
Added December 10, 2013
1 Communities
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11 MeSH Terms