Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family

“…Enzyme activities were measured at D 280 0.05-0.2 OU. Concentrations of papain, ficin, cathepsins B and L were determined as described in Semashko et al (2014), using the titration of the active centers of enzymes by E-64, an irreversible inhibitor of cysteine peptidases. Trypsin concentration was similarly evaluated by data on titration of active sites.…”

“…The initial rates of hydrolysis of the chromogenic substrates were determined according to (Semashko et al, 2014) by similar procedure as described above with substrate concentrations of 0.05 -0.5 mM. The ratio k cat /K M was calculated from the values of kinetic parameters obtained by nonlinear regression using the program OriginPro 7.5 or by linearization of the standard Michaelis-Menten equation by Hanes-Woolf and Lineweaver-Burk plots (Dixon and Webb, 1982) using Microsoft Excel.…”

“…The resulting compounds Glp-Phe-Gln-pNA and Glp-Phe-Gln-AMC were compared to alanine-containing selective substrates, Glp-Phe-Ala-pNA and Glp-Phe-Ala-AMC, that were synthesized and described in our laboratory earlier (Semashko et al, 2014), in terms of their hydrolysis efficiency by the studied cysteine peptidases. Two parameters were compared: (1) specific activity (M substrate /M enzyme * min) ( Table 1) and (2) specificity constant k cat /K M ( Table 2) which characterizes both the rate of the enzymatic reaction (k cat value) and the affinity of a given enzyme to a given substrate (K M value).…”

“…Cysteine cathepsins are not only lysosomal peptidases, as there is clear evidence of their localization in other cellular compartments, such as the nucleus, cytoplasm and plasma membranes (Wex et al, 2003;Hsing and Rudensky, 2005;Salminen-Mankonen et al, 2007). Of particular interest are the cysteine cathepsins found in the multi-enzyme digestive complex of some insects (Vinokurov et al, 2005(Vinokurov et al, , 2006a(Vinokurov et al, ,b, 2009Semashko et al, 2014), as well as carnivorous plants (Ravee et al, 2018). Being digestive, these cathepsins not only have a higher degradomic potential than ordinary lysosomal cathepsins because of expanded substrates, but due to the structure of their natural substrates, they also exhibit specificity that is not characteristic of most known lysosomal cathepsins.…”

An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides

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ВВЕДЕНИЕХромогенные субстраты ферментов часто используются для определения активностей последних с помощью фотометрических методов [1,2]. Так как данные методы широко применяются в научных исследованиях, клинической диагностике, при проведении анализов в биотехнологической промышленности, экологии и иных областях для выявления ферментов и оценки их активности, потребности в специфичных хромогенных субстратах весьма высоки, и многие из этих субстратов есть в каталогах зарубежных компаний -производителей и поставщиков реактивов.Тромбин, он же фактор свёртывания II, играет одну из ключевых ролей в процессе свёртывания крови.

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