Chloromethylketone EGR (EGRCK) (Biotin), Purified

EGRCK labeled with biotin is prepared by the method of Williams, et al. (11). Tri-peptide chloromethylketones have been utilized extensively to irreversibly inhibit various serine proteases (1-5). Among the most common chloromethylketones are FPRCK (Phe-Pro-Arg-chloromethylketone), which is a rapid inhibitor of a-thrombin and EGRCK (Glu-Gly-Arg-chloromethylketone), which rapidly inhibits factor Xa (1). Recently, the modification of these tri-peptide chloromethylketones with reporting groups, such as fluorescent probes (6-8), radioactive labels (9) or thioreactive-labels (10), has provided a unique approach to the study of various serine proteases. These probes are useful because they allow a means of reporting molecular changes in an enzyme, and not its zymogen, while also inhibiting the enzymatic activity. , The use of biotin as a reporting group has been used extensively with antibodies in ELISA based assays and in Western Blot. The biotin, in conjunction with avidin, creates a highly sensitive method for detecting antibodies, and therefore, antigens. By modifying the tripeptide-chloromethylketones with a biotin group, the sensitivity of the avidin/biotin system can be extended to study serine proteases without the need for specific antibodies to the active enzymes, Applications: Biotinylated tripeptide chloromethyl ketones can be used in a variety of ways (11-13). First, the compounds can be reacted with unwanted serine proteases in a sample or preparation, and can then be removed along with the protease using avidin-Sepharose (11). Second, the biotinylated-serine protease can be visualized on a blot without the use of specific antibodies (11). Third, the biotinylated serine protease can be quantitated in an active-site specific immunoassay (12,13). The spacer utilized on these compounds has been optimized to allow good reactivity of the biotinylated FPRCK and the biotinylated EGRCK in the above mentioned procedures. , In addition to biotinylated chloromethyl ketones, fluorescein labeled compounds are also available. The fluorescein labeled compounds are useful in both Western blot and fluorescent imaging applications. Additional Specifications: Special Properties: Tri-peptide chloromethyl ketones are very potent and irreversible inhibitors of serine proteases. BEGRCK is useful for inhibition of factor Xa. The biotin moiety provides the ability to use the peptide-chloromethyl ketones as specific probes for detection and/or capture of serine proteases via the avidin/biotin interaction. Storage and Stability: Store at -20°C or lower. Aliquots are stable for at least 6 months at -20°C or lower. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.