| 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 -20C or lower. Aliquots are stable for at least 6 months at -20C or lower. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
