Submitted to: Analytical Biochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/23/2013
Publication Date: 3/15/2014
Publication URL: http://handle.nal.usda.gov/10113/58815
Citation: Semashko, T.A., Vorotnikova, E.A., Sharikova, V.F., Vinokurov, K.S., Smirnova, Y.A., Dunaevsky, Y.E., Belozersky, M.A., Oppert, B.S., Elpidina, E.N., Filippova, I.Y. 2014. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures. Analytical Biochemistry. 449: 179-187. doi: http://dx.doi.org/10.1016/j.ab.2013.12.032. Interpretive Summary: Cysteine peptidases are enzymes that are critical to most life processes. Important to our research, these peptidases are important in the digestion of protein in many stored product beetles. The currently available commercial substrates used to analyze these enzymes have problems, including low solubility, lack of sensitivity and selectivity, and spontaneous hydrolysis. Therefore, we designed new substrates for cysteine peptidases that address many of these problems. The new substrates were demonstrated to have improved solubility and were selective for a specific group of cysteine peptidases. These substrates were superior in the analysis of cysteine peptidases in stored product beetles. The new substrates will be useful in cases where specificity is needed in the analysis of cysteine peptidases in complex systems.
Technical Abstract: Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-Y, where Glp – pyroglutamyl, Y = pNA (p-nitroanilide), AMC (4-amino-7-methylcoumaride) or AFC (4-amino-7-trifluoromethyl-coumaride); Xaa = Phe, Val. The synthesis of substrates was enzymatic to guarantee the selectivity of the reaction and provided optical purity of the target compounds, thus simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was studied by representative cysteine peptidases from the C1 family: the plant enzymes papain, bromelain, ficin; and bovine and human lysosomal cathepsins B and L, respectively. The synthesized substrates were selective for cysteine peptidases of the C1 family and were not hydrolyzed by peptidases of other clans: serine peptidases - trypsin, a-chymotrypsin, subtilisin Carlsberg; the aspartic peptidase pepsin; and the metallopeptidase thermolysin. The substrates were successfully used to monitor the activities of cysteine peptidases during the chromatographic separation of a multi-component set of digestive peptidases from a beetle, Tenebrio molitor. The synthesized substrates and selective inhibitors were used to characterize cysteine peptidases in multi-component mixtures of midgut extracts from T. molitor larvae as well as larvae of beetles from the genus Tribolium. These studies demonstrate that the newly synthesized substrates are selective for C1 cysteine peptidases and are useful in the analysis of complex mixtures containing cysteine peptidase enzymes.