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目的：建立兔乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）分离纯化的新方法，研究该酶的动力学特性。方法：采用ＢｌｕｅＤｅｘｔｒａｎ和Ｓｅｐｈａｒｏｓｅ４Ｂ交联制成ＢｌｕｅＤｅｘｔｒａｎ－Ｓｅｐｈａｒｏｓｅ４Ｂ染料配体亲和层析柱，结合ＱＡＥ－ＳｅｐｈａｄｅｘＡ５０离子交换层析分离提纯兔ＬＤＨ－Ｃ４，以作图法测定该酶动力学数据。结果：纯化的ＬＤＨ－Ｃ４经ＳＤＳ－ＰＡＧＥ及琼脂糖凝胶电泳证明为单一组分，相对分子质量１３６０００。纯化得率４５．５％，纯化倍数３１６倍。兔ＬＤＨ－Ｃ４动力学测定，其对底物乳酸、丙酮酸的Ｋｍ值分别为１．１１ｍｍｏｌ／Ｌ、０．２３ｍｍｏｌ／Ｌ；产物乳酸、丙酮酸对其均有非竞争性抑制作用，Ｋｉ值分别为９．１１ｍｍｏｌ／Ｌ、０．０２７ｍｍｏｌ／Ｌ。结论：本法是一种简便、快速、高效的分离纯化免ＬＤＨ－Ｃ４新方法；动力学性质表明该酶对丙酮酸的亲和力大于乳酸，且易受产物丙酮酸的抑制。
OBJECTIVE: To establish a new method for the isolation and purification of rabbit lactate dehydrogenase C4 (LDH-C4) and to study its kinetic properties. METHODS: BlueDextran-Sepharose 4B dye affinity chromatography column was prepared by using BlueDextran and Sepharose 4B cross-linking. Rabbit LDH-C4 was separated and purified by QAE-SephadexA50 ion exchange chromatography. The enzyme kinetics data were determined by the method of mapping. Results: The purified LDH-C4 was proved to be a single component by SDS-PAGE and agarose gel electrophoresis. The relative molecular mass was 136000. Purification rate 45.5%, purification multiple 316 times. Rabbit LDH-C4 kinetic assay, the substrate of lactic acid, pyruvate Km values were 1.11mmol / L, 0.23mmol / L; product lactate, pyruvate non-competitive inhibition of both, Ki Respectively, 9.11mmol / L, 0.027mmol / L. Conclusion: This method is a simple, rapid and efficient method for isolation and purification of free LDH-C4. Its kinetic properties indicate that the enzyme has higher affinity for pyruvate than lactic acid and is susceptible to pyruvate production.