ACTIVE SITE-SELECTIVE LABELING OF THROMBIN WITH FLUORESCENCE PROBES USING THI0ESTER DERIVATIVES OF PEPTIDE-CHL0R0METHYL KETONES

Abstract

Active site-directed inactivation of a serine protease with a thioester derivative of a peptide-chloromethyl ketone followed by reaction of the unique thiol group generatedin the presence of hydroxylamine with a fluorophore-iodoacetamide hasbeen investigated as a new method for covalent incorporation of extrinsic fluorescence probes into the active sites of blood coagulation proteases. The specificity of labeling by this method was evaluated by quantitation of the reactions between human thrombin, acetylthioacetyl-D-Phe-Pro-ArgCH2Cl (ATA-FPRCK) and 5-iodoacetamidofluorescein(IAF).ATA-FPRCK was synthesized by reaction of FPRCK with succinimidylacetylthioacetate and purified by chromatography on SP-Sephadex and Sephadex G10. Titrations of the loss of thrombin chromogenic substrate activity with ATA-FPRCK were linear, with end points of 1.1-1.2 mol ATA-FPRCK added/mol active sites, consistent with a reaction stoichiometry of 1 and the ∽90% purity of the compound estimated by reverse-phase HPLC.Inactivation of thrombin wasquantitatively correlated with incorporation of the thioester, with a maximum of 1.04 mol/mol active sites.IAF labeling of ATA-FPR-thrombin inthe presence of 0.1M NH20H yielded a maximu of 0.96 mol IAF incorporated/mol active sites in a reaction accompanied by loss of the thiol group. Incorporation of ATA-FPRCK wasdependent on thefunctional thrombin active site, asdemonstrated by less than 4%thioester or IAF incorporation for the enzyme previously inactivated with FPRCK. I conclude that active site-selective fluorescence labelingcan be achieved by the method described here with the advantage of a wide choice in the properties of theprobe incorporated. In addition, a 2.3-fold difference in fluorescenceintensity was observed for 2,6-ANS derivatives of ATA-FPR-thrombin andATA-D-Phe-Phe-Arg-thrombin, indicating that the spectral properties ofenvironmentally sensitive fluorescence probes are influenced by the structure of the peptide inhibitor.Supported in part by a grant from the American National Red Cross.