Guoth J G, Sohar I, Zarandi M, Janaky T, Schally A V
Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, New Orleans, Louisiana 70146.
Prep Biochem. 1988;18(3):361-74. doi: 10.1080/00327488808062534.
Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.
使用匀浆、超速离心、高效水相尺寸排阻色谱和高效阴离子交换色谱法，从大鼠肌肉提取物中纯化出两种MMP-7酶同工酶，纯化了100倍，达到明显的均一性，总产率为10%。使用TSK G-2000SW柱时，分离使同工酶B和C得到6倍的纯化和30%的回收率。然后将这种浓缩的酶提取物通过TSK-DEAE-2SW柱，在pH 7.5下使用盐梯度，使同工酶进一步纯化25倍，回收率为90%。在阴离子交换器和反相HPLC柱上对活性酶进行纯度测试时，检测到代表同工酶B和C的两个对称酶峰。所涉及的步骤包括提取、超速离心、色谱分析和酶分析，耗时不到5小时。
