Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

P.E Bock

doi:10.1016/s0021-9258(18)42134-5

Abstract

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%.

Highlights

Active-site-selective Labelingof Blood Coagulation Proteinases with Fluorescence Probes by the Use of Thioester Peptide Chloromethyl Ketones
This approach was de- niques employing blood coagulation enzymes that have been veloped to increase the opportunities for identifying covalently labeled withextrinsic fluorescence probes have fluorescent proteinase derivativesthat act as reporters been used to quantitate andcharacterize some of these interof binding interactions by allowing a large number of actions, thoseregulating the formation and activderivatives, representing a broad rangofe probe spec- ity of thrombin [2,3,4,5,6,7]
Subsequent mild treatment of the proteins modified with fluorescence probes, such as actin [9], covalent thrombin-inhibitor complexeswith NH20Hin indicate that different probes incorporated at the same site the presence of 5-(iodoacetamido)fluoresceinresulted may provide information characteristicof each probe by prefin generation of the thiol group together witihts selec- erentiallyreporting different aspects of the process under tive modification and incorporation of 0.96 f 0.07 mol investigation

Summary

SCHEMI E

The rates of hydrolysis were too slow to result in significant decreases in the inhibitor concentration on the time scale of unique site for selective chemical modification with thiol- the kinetic studies. CH&l showed incorporation of less than 0.04 mol of thioes- reactions of the thioester peptide chloromethyl ketones with ter/mol of active sites, demonstratingan essentially complete thrombin accompanying the first step of Scheme I were evaldependence of incorporation onthe presence of the functional uated. Nonspecific acetylation was quantitated from the amount of thiol generated over 20-27 h, measuredby the absorbance increaseat 412 nm accompanyingthe DTNB reaction and correctedfor blanks of the proteins and inhibitors incubatedwithDTNBseparately.Nonspecificalkylation of prethrombin 1 andprothrombinin the same incubations was determined after dialysis of the proteins from the amount of thioester incorporated,measured as describedunder “ExDerimental Procedures.” Pooled results are listed from two experiments inwhich thrombin (10-25 p M ) , prethrombin 1 (16-25 pM), or prothrombin(24-48 PM)was incubated with 100 PM inhibitor and 480 p~ DTNB in pH 7 buffer at 25 “C. Nonspecific acetylation was quantitated from the amount of thiol generated over 20-27 h, measuredby the absorbance increaseat 412 nm accompanyingthe DTNB reaction and correctedfor blanks of the proteins and inhibitors incubatedwithDTNBseparately.Nonspecificalkylation of prethrombin 1 andprothrombinin the same incubations was determined after dialysis of the proteins from the amount of thioester incorporated,measured as describedunder “ExDerimental Procedures.”

Thioester incorporated rnolfrnol protein

DISCUSSION

Erntuo Punficalmn and Charam