Comparison And Development Of Measuring Methods For Pyruvate Dehydrogenase Complex

Pyruvate dehydrogenase complex is a key enzyme which plays a pivotal role in energy and physical metabolism. For most of the studies on pyruvate dehydrogenase (or pyruvate dehydrogenase complex) such as its configuration and property investigation, gene cloning, separation, purification and identification etc, it is necessary and has highly academic significance to measure the enzymatic activity. At the same time, it has highly practical significance for investigations which involve disorders of pyruvate metabolism and filtration and exploitation of herbicide or bactericide to think of PDH as target.At present, the assay methods for the pyruvate dehydrogenase activity that has been reported were almost developed in 1950-60s. According to the technologies it takes, the enzyme activity detecting approaches could be divided into spectrophotometric and radiochemical assays. Radiochemical assay has mainly been used to measuring PDH (or PDHc) activity in crude homogenates based on the cofactor-dependent conversion of [^1-14C]pyruvate to ¹⁴CO₂ to reflect enzymatic activity. Spectrophotometric assays for PDH_C contain conventional standard spectrophotometric assay based on measuring the rate of formation of NADH and an INT-coupled assay. Two most popular spectrophotometric approaches for the measurement of purified El activity are the strategies based on reaction with ferricyanide and 2,6-dichlorophenolindophenol (2,6-DCPIP).The kinetic measurements are more complicated in radiochemical assay than those in spectroscopic procedures. Simultaneously, unstable radioactive substrates which result in environmental contamination will be used in this method. So this investigation set emphases in spectrophotometric assay, using the same one method to measure different enzymatic activities and different methods to measure the same one enzymatic activities, we had studied different measurement method about its sensitivity, disturbing factor and so on. Simultaneously, in the thesis a new optimized spectrophotometric assay for measurement of PDH's activity was put forward firstly.By systemic studies to test assays commonly used for pyruvate dehydrogenase (or pyruvate dehydrogenase complex), It was found that the sensitivity of method based on reaction with ferricyanide was very poor and the method involving reaction with 2,6-DCPIP might not reflect the exactly enzymatic activity on account of some bad interferences of sulfhydryl group-protecting reagents. Conventional standard spectrophotometric assay based on measuring the rate of formation of NADH is used for the measuring of PDHc activity in purified pyruvate dehydrogenase complex. The method was limited by feedback inhibition on the PDH_C reaction and agents were expensive. In 1981, an INT-coupled assay is applied to estimate the PDHc activity in crude homogenates by Hinman L M.. According to the studies in this thesis, we should pay attention to reliability on the activity measured by INT-coupled assay because INT that be supposed to react with NADH of product of PDH_C may not only react with NADH but also direct react with product of E1.In the present paper we have endeavored to develop a new, effective and economical method for the measurement of PDH activity for all sorts of disadvantages of previous methods. The principle of the method is base on measurement of reduced MTT induced by the product of PDH and the highest point of absorbance moves from 420nm (yellow) to 570nm (blue). MTT works as electron acceptor in this method. The sensitivity of new method is much higher than that of 2, 6-DCPIP assay and ferricyanide assay and one advantage of the PMS-MTT assay was the low cost of the reagents compared with standard NADH assay. Further more, this assay has good stability to avoid the interference of protecting reagent to enzyme activity. This MTT-coupling assay is applicable to measure the activities of PDH in both purified pyruvate dehydrogenase and pyruvate dehydrogenase in PDH_C isolated mitochondria samples.