El Hakim A E, Salama W H, Hamed M B, Ali A A, Ibrahim N M
Molecular Biology Department, National Research Centre, 33 Bohouth St. (former El Tahrir St.), Dokki, Giza, Egypt.
Durham University, School of Biological and Biomedical Sciences, Durham DH1 3LE, United Kingdom.
J Genet Eng Biotechnol. 2015 Dec;13(2):165-176. doi: 10.1016/j.jgeb.2015.09.003. Epub 2015 Sep 26.
Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The and values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn ion markedly increased the LAAO activity for both purified isoforms, while Na, K, Ca , Mg and Ba ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn , Ni , Co , Cu and AL ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.
通过包括凝胶过滤和阴离子交换色谱在内的连续两步色谱方法，从毒液中纯化出两种L-氨基酸氧化酶同工型，即Cc-LAAOI和Cc-LAAOII，达到了表观均一性。通过在已校准的Sephacryl S-200柱上进行凝胶过滤测定，这些同工型的天然分子量为115 kDa，而LAAOI和LAAOII酶的单体分子量分别为60、56 kDa和60、53 kDa。这两种同工型的胰蛋白酶肽与其他蛇毒L-氨基酸氧化酶具有高度的序列同源性。Cc-LAAOI和Cc-LAAOII的最佳pH值和温度值分别为7.8、50°C和7、60°C。这两种同工酶在高达70°C时具有热稳定性。LAAOI的Km和Vmax值分别为0.67 mM、0.135 μmol/min，LAAOII的Km和Vmax值分别为0.82 mM、0.087 μmol/min。两种同工酶对长链、疏水和芳香族氨基酸均表现出较高的催化偏好性。锰离子显著提高了两种纯化同工型的LAAO活性，而钠、钾、钙、镁和钡离子对两种同工型的酶活性增加不显著。此外，锌、镍、钴、铜和铝离子显著抑制LAAOI和LAAOII的活性。L-半胱氨酸和还原型谷胱甘肽完全抑制了两种同工酶的LAAO活性，而β-巯基乙醇、邻菲罗啉和苯甲基磺酰氟完全抑制了LAAOII的酶活性。此外，碘乙酸抑制LAAOII的酶活性达46%，对LAAOI活性无影响。
