Table_1_New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family.DOCX

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.

本研究针对C1木瓜蛋白酶家族（C1 papain family）肽酶的活性检测，开发了一类P1位（P1-position）含谷氨酰胺的新型底物，其通式为Glp-Phe-Gln-X，其中Glp为焦谷氨酰基（pyroglutamyl），X为pNA（对硝基苯胺，p-nitroanilide）或AMC（4-氨基-7-甲基香豆素，4-amino-7-methylcoumaride）。该类底物结构简洁，可被多种来源的C1家族半胱氨酸肽酶（cysteine peptidases）高效切割。这类底物的核心优势在于对C1家族半胱氨酸肽酶的高选择性：其他肽酶类群（包括丝氨酸胰蛋白酶样肽酶（serine trypsin-like peptidases））均无法裂解含谷氨酰胺的此类底物。本研究证实，将Glp-Phe-Gln-pNA与商业化底物Z-Arg-Arg-pNA联用，可实现组织蛋白酶L与B（cathepsins L and B）的差异化鉴别。相较于此前报道的含丙氨酸的原型底物，本研究提出的新型底物在比活性（specific activity）与动力学参数（kinetic parameters）方面均更具优势。通过对拟步甲科（Tenebrionidae）储粮害虫的多酶消化复合物开展色谱与电泳分析，本研究验证了该类底物的高效性与选择性。