Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases.
Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases.
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DOI：
10.1016/j.ab.2011.06.031
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发表时间：
2011-11-01
影响因子：
2.9
通讯作者：
Silverman DN
中科院分区：
文献类型：
--
作者：
Moral ME;Tu C;Richards NG;Silverman DN
膜进样质谱法（MIMS）使用穿过渗透膜的扩散来检测溶液中不带电荷的小分子量分子。本文以枯草芽孢杆菌草酸脱羧酶（OxDC）为例，介绍了MIMS在测定脱羧酶催化特性中的应用。脱羧酶活性从非氧化反应产生二氧化碳和甲酸盐，但伴随着消耗草酸盐和氧并产生CO2和过氧化氢的伴随氧化酶活性。MIMS在OxDC催化测量中的应用包括从各自质量峰的离子电流中实时和连续地检测氧气和产物CO2。通过测量产物CO2使用MIMS获得的脱羧酶活性的稳态催化常数与通过基于与甲酸脱氢酶的偶联反应的传统终点测定获得的那些相当，并且测量O2的消耗使用MIMS也估计氧化酶活性。在MIMS中使用同位素标记的底物（富含13 C2的草酸盐）提供了一种通过检测产物13 CO2（m/z 45）的质量峰来表征细胞悬浮液中的催化反应的方法，避免了由于内源性12 CO2引起的不准确性。
Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the non-oxidative reaction, but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO2 and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO2 from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO2 using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O2 using MIMS also estimates the oxidase activity. Use of isotope-labeled substrate (13C2-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product 13CO2 (m/z 45), avoiding inaccuracies due to endogenous 12CO2.
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