Research on the development of highly sensetive methods for trace metals in biological fluids

建立了测定生物体液中钙离子的酶促分析方法。测定原理是用胆碱氧化酶和过氧化物酶测定被钙离子激活的磷脂酶D的活性。我发现，虽然发色链霉菌的磷脂酶D在很窄的钙浓度范围内被激活，但当在反应混合物中加入除钙离子外的二价金属离子时，激活磷脂酶D的钙离子浓度范围扩大。在二价以上的金属离子中，Mn~(2+)&gt~(2+)的效率最高，在含有Mn~(2+)和gt~(2+)的反应混合物中，磷脂酶D的反应速率随20 mM Ca~(2+)&gt；的浓度呈线性增加。钙的测定不受其他离子的影响，如镁离子和钙离子。本方法测定血清钙的结果与原子吸收光谱法测定的结果一致。用化学发光法和由磷脂酶D和胆碱氧化酶组成的生物反应器对钙进行了高灵敏度的分析。用乙醇脱氢酶(ALDH)和碱性磷酸酶(ALP)等锌酶，尝试用酶法测定锌。虽然在脱锌的apo-ALDH中加入了锌溶液，但没有再次显示出活性。络合剂脱锌的apo-ALP活性随锌浓度的增加而增加。热裂解酶分子中含有1摩尔的锌，它的锌很容易被络合剂除去。但由于热裂解酶是一种蛋白水解酶，其活性测定比较困难。

The development of the enzymatic method for determining Ca^<2+> in biological fluids was trid. The assay principle was based on determining the activity of phospholipase D activated by Ca^<2+> using choline oxidase and peroxidase. I found that though phospholipase D from Streptomyces chromofuscus was activated in the very narrow range of the Ca concentration, the range of the Ca^<2+> concentration activating phospholipase D expanded, when a divalent metal ion except Ca^<2+> was added to the reaction mixture. Mn^<2+> among the metal ions above divalent showed the best efficiency, and the reaction rate of phospholipase D increased linearly with the concentration of 20 mM Ca^<2+> in the reaction mixture containing Mn^<2+>. The Ca assay was not influenced with other ions, such as Mg^<2+>. The results of serum Ca by the present method correlated with those by the atomic absorption spectrometry. The highly sensitive analysis of Ca was tried using chemiluminescence and the bioreactor consisting of phospholipase D and choline oxidase. It detected 10 pmol H_2O_2 per muL sample.The enzymatic method for determining Zn wa tried using the Zn-enzyme such as alcohol dehydrogenase (ALDH) and alkaline phosphatase (ALP) . Though a Zn solution was added to the apo-ALDH removed Zn, it did not show the activity again. The activity of the apo-ALP removed Zn by a chelate agent increased with the concentration of Zn added. Thermolysin had 1 mol Zn in the molecule, and its Zn was easily removed by a chelate agent. But because thermolysin was a kind of protease, the determination of its activity was difficult.