Allen R E, Lo T W, Thornalley P J
Department of Chemistry and Biological Chemistry, University of Essex, Wivenhoe Park, Colchester, United Kingdom.
J Protein Chem. 1993 Apr;12(2):111-9. doi: 10.1007/BF01026032.
Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. The KM value for methylglyoxal-glutathione hemithioacetal was 192 +/- 8 microM and the kcat value was 10.9 +/- 0.2 x 10(4) min-1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had a Ki value of 0.16 +/- 0.04 microM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with a Ki value of 3.12 +/- 0.88 microM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with a Ki value of 18 +/- 8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.
乙二醛酶I（EC 4.4.1.5）通过一种简化方法从人红细胞中纯化得到，该方法使用S-己基谷胱甘肽亲和色谱，并采用改良的S-己基谷胱甘肽浓度梯度进行洗脱。纯蛋白的比活性为1830 U/mg蛋白，总产率为9%。纯蛋白的分子量为46,000 D，由两个各为23,000 D的亚基组成，等电点值为5.1。甲基乙二醛-谷胱甘肽半硫代缩醛的KM值为192±8 μM，kcat值为10.9±0.2×10⁴ min⁻¹（N = 15）。乙二醛酶I抑制剂S-对溴苄基谷胱甘肽的Ki值为0.16±0.04 μM，而之前认为仅抑制乙二醛酶II的S-对硝基苄氧基羰基谷胱甘肽也能抑制乙二醛酶I，其Ki值为3.12±0.88 μM。还原型谷胱甘肽是乙二醛酶I的弱竞争性抑制剂，Ki值为18±8 mM。用纯化的酶制备了多克隆抗体，通过免疫印迹发现其能与乙二醛酶I抗原特异性反应。该方法通过简单的低压色谱技术得到了高纯度的蛋白，对于小规模制备而言产率适中但足够。
