The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.
The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.
复制标题
通过氚损失法估算的同位素效应对脯氨酸脱氢速率的影响。
DOI：
10.1016/0003-2697(92)90302-n
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发表时间：
1992
影响因子：
2.9
通讯作者：
Kohl,DH
中科院分区：
文献类型：
--
作者：
Shearer,G;Jones,JR;Kohl,DH
氚从基质中的损失通常用于估计脱氢速率。然而，由于氚同位素效应，3 H的损失可能比H的损失慢得多。为了评估氚同位素效应的影响，在大鼠肝线粒体和来自大豆（Glycine max [L.]梅里尔）结节与[14 C]脯氨酸脱氢产物中14 C的出现进行了比较。在存在邻氨基苯甲醛（添加以捕获初始产物Δ1-吡咯啉-5-羧酸酯）的情况下进行孵育。邻氨基苯甲醛捕获的总14 C产物的分数从0.07到0.75不等，这取决于实验条件。与大鼠肝线粒体，[14 C]脯氨酸脱氢是3.27和9.25倍之间的3 H脯氨酸脱氢快，这取决于测定条件。大豆根瘤类杆菌使[14 C]脯氨酸脱氢的速度比[3 H]脯氨酸快约5倍。我们得出以下结论：（i）由于氚同位素效应，氚测定法可能大大低估脯氨酸脱氢的速率;（ii）如果假定邻氨基苯甲醛在所有条件下都能定量捕获Δ1-吡咯啉-5-羧酸，则14 C测定法可能低估脯氨酸脱氢的速率。氚含量测定的简单性使其对常规使用具有吸引力。然而，其使用需要在特定的分析条件下测定氚同位素效应，以校正结果。这里讨论的考虑因素对任何使用氚损失的脱氢酶测定具有广泛的适用性。
Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, Δ1-pyroline-5-carboxylate). The fraction of total14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of3H proline, depending upon assay conditions. Soybean nodule bacteroids dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps Δ1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.
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作者：
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作者：
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DOI：
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