McNevin Dennis B, Badger Murray R, Kane Heather J, Farquhar Graham D
Molecular Plant Physiology, Research School of Biological Sciences, The Australian National University, Canberra, 0200 ACT, Australia.
Environmental Biology, Research School of Biological Sciences, The Australian National University, Canberra, 0200 ACT, Australia.
Funct Plant Biol. 2006 Dec;33(12):1115-1128. doi: 10.1071/FP06201.
Methods for determining carbon isotope discrimination, Δ, or kinetic isotope effects, α, for CO-consuming enzymes have traditionally been cumbersome and time-consuming, requiring careful isolation of substrates and products and conversion of these to CO for measurement of isotope ratio by mass spectrometry (MS). An equation originally derived by Rayleigh in 1896 has been used more recently to good effect as it only requires measurement of substrate concentrations and isotope ratios. For carboxylation reactions such as those catalysed by d-ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, EC 4.1.1.39) and PEP carboxylase (PEPC, EC 4.1.1.31), this has still required sampling of reactions at various states of completion and conversion of all inorganic carbon to CO, as well as determining the amount of substrate consumed. We introduce a new method of membrane inlet MS which can be used to continuously monitor individual CO isotope concentrations, rather than isotope ratio. This enables the use of a simplified, new formula for calculating kinetic isotope effects, based on the assumptions underlying the original Rayleigh fractionation equation and given by.
传统上，测定消耗 CO 的酶的碳同位素分馏（Δ）或动力学同位素效应（α）的方法既繁琐又耗时，需要仔细分离底物和产物，并将它们转化为 CO，以便通过质谱（MS）测量同位素比率。1896 年瑞利最初推导的一个方程最近得到了很好的应用，因为它只需要测量底物浓度和同位素比率。对于羧化反应，如由 1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO，EC 4.1.1.39）和磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31）催化的反应，这仍然需要在反应的不同完成状态下取样，并将所有无机碳转化为 CO，以及确定消耗的底物量。我们介绍了一种新的膜进样质谱方法，该方法可用于连续监测单个 CO 同位素浓度，而不是同位素比率。这使得基于原始瑞利分馏方程的假设并由给出的用于计算动力学同位素效应的简化新公式得以使用。
