A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity
A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity
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DOI：
10.1016/j.ab.2005.09.027
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发表时间：
2005-12-15
影响因子：
2.9
通讯作者：
Hung, HC
中科院分区：
文献类型：
--
作者：
Liao, YF;Hsieh, HC;Hung, HC
介绍了一种简单的连续分光光度法测定肽精氨酸脱亚胺酶（PAD）的方法。肽基精氨酸的脱亚反应产生肽基瓜氨酸和氨。肽精氨酸水解过程中释放的氨与谷氨酸脱氢酶催化的α -酮戊二酸还原胺化成谷氨酸和还原的烟酰胺腺嘌呤二核苷酸（NADH）氧化相结合。连续测量了在340 nm处NADH氧化引起的吸光度消失。通过这种新方案获得的高纯度人PAD的比活性与常用的比色法获得的活性相当，该比色法通过偶联双乙酰单肟来测量肽基瓜氨酸的尿醛基。该方法灵敏度高，准确度高，适用于PAD的酶动力学分析。通过这种方法获得的PAD半最大活性的Ca2+浓度与以前通过比色法获得的浓度相当。(c) 2005爱思唯尔公司版权所有。
A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340 nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca2+ concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure. (c) 2005 Elsevier Inc. All rights reserved.