Optimal Conditions for Kinetic Study of Succinate Dehydrogenase in Rat Liver

Journal of the Arkansas Academy of Science Volume 40 Article 21 1986 Optimal Conditions for Kinetic Study of Succinate Dehydrogenase in Rat Liver Collie B. Shaw University of Central Arkansas Tara L. Chronister University of Central Arkansas John D. Peck University of Central Arkansas Follow this and additional works at: http://scholarworks.uark.edu/jaas Part of the Molecular Biology Commons Recommended Citation Shaw, Collie B.; Chronister, Tara L.; and Peck, John D. (1986) "Optimal Conditions for Kinetic Study of Succinate Dehydrogenase in Rat Liver," Journal of the Arkansas Academy of Science: Vol. 40 , Article 21. Available at: http://scholarworks.uark.edu/jaas/vol40/iss1/21 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact , . Journal of the Arkansas Academy of Science, Vol. 40 [1986], Art. 21 OPTIMAL CONDITIONS FOR KINETIC STUDY OF SUCCINATE DEHYDROGENASE INRAT LIVER COLLIE B. SHAW, TARA L. CHRONISTER* and JOHN D. PECK Department of Biology University of Central Arkansas Conway, AR 72032 ABSTRACT Succinate dehydrogenase (SDH) commonly is assayed as a marker enzyme for mitochondrial activity. The literature presents numerous conditions for conducting this assay due to the fact that, ithas been difficult to get sufficient reduction of the acceptor dye, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). This study was undertaken to optimize the SDH-catalyzed reduction of TTC dye by evaluation of a greater range of molor ratios of TTC to succinate and by further evaluation of additives reported as beneficial. Improvement in enzyme specific activity was achieved by liver perfusion via the left cardiac ventricle with homogenizing solution. Increase inTTC from 1 to 1 0 mM and further increase to 20 mM resulted in major improvement in color production. The greatest improvement in apparent activity was achieved by addition of 1 mM phenozine methosulfate, a hydrogen transfer mediator. Use of CaCI2l EDTA, Triton X-100, NaN 3 and KCN was not beneficial. The above modifications of the SDH assay resulted ingreater sensitivity, the conduct of a greater number of assays with less tissue and the sacrifice of fewer animals. INTRODUCTION Succinate dehydrogenase (SDH) commonly is used as a marker enzyme for the mitochondrion because it is bound to the inner membrane of the mitochondrion. Frequently, its activity is used to assess tricarboxylic acid cycle activity as wellas electron and hydrogen transfer to the electron transport system. The activity of SDH usually is measured spectrophotometrically by following the reduction of an artificial acceptor dye such as 2,3,5-triphenyI-2H-tetrazolium chloride (TTC). Numerous conditions have been presented in the literature for conducting this assay, due to difficulty inobtaining sufficient dye reduction and adequate absorbance values when using reasonable amounts of enzyme. For determination of Michaelis-Menton constants for inhibitedand uninhibited reactions catalyzed by SDH, itis important to obtain high absorbance values for the uninhibited reaction. Otherwise inhibitors may decrease the formation of reduced dye and absorbance values to such a point that instrumental error becomes too great to provide reliable measurements. This study was initiated to optimize the SDH catalyzed reduction of TTC dye by evaluating a greater range of molar ratios of succinate to TTC dye and by further evaluation of additives mentioned in the literature. These include EDTA, a chelator, CaCh and Triton X-100, substances reputed to increase membrane permeability, KCNand NaN,, electron transport inhibitors, and phenazine methosulfate, a putative electron transfer mediator between FADH2 and TTC. This paper presents improvements inthe assay that willincrease the SDH catalyzed reduction of TTC, give high dye absorbance values for uninhibited reactions and adequate absorbance for inhibited reactions. This will facilitate future kinetic studies of SDH. MATERIALS AND METHODS Chemicals used in this study were obtained from 4 major chemical companies. 2,3,5-triphenyl-2H-tetrazolium chloride monohydrate (TTC) and sodium dithionite were obtained from Aldrich Chemical Company. Phenazine methosulfate, sodium azide, and sucrose were obtained from Sigma Chemical Company. Disodium succinate was obtained from National Biochemical Corporation. Sodium phosphate, ether, acetone, and potassium cyanide were obtained from Fisher Scientific Company. The necessary aqueous solutions were made with distilled, deionized water. The animals, black-hooded derived rats, were housed in environmen- tally controlled conditions and were provided food and water ad libitum. The animals were sacrificed by stunning and cervical dislocation or by etherization, and exsanguination. Exsanguination was achieved by perfusion with ice-ccld homogenizing solution (.25 Msucrose) vialeft cardiac ventricle until the liver became more lightly colored. The liver was excised, weighed, placed in a 0°-5°C solution of homogenizing fluid, diced and transferred to a prechilled homogenizing vessel. Homogenization was achieved by 5 passes of a teflon pestle into a glass vessel (size C Thomas). The homogenize r was powered by a Talboys Instrument Corporation Model 102 electric motor operated at full speed. The homogenate was maintained at less than 5°C during this procedure. The homogenate was then placed ina refrigerated Sorvall RC2-B centrifuge and spun at 800 xG for 10 minutes. The supernatant (SI) was collected, diluted with 0.25 Msucrose and recentrifuged at 20,000 xG for 20 minutes to pellet the mitochondria. The supernatant (S2) was removed and the pellet (P2) was resuspended in .25 M sucrose in the ratio of 1-2 gm wet weight of liver to 1 ml sucrose. This suspension was centrifuged at 600 xG for 5 minutes to remove all large particles not resuspended in the earlier step. The supernatant (S3) was kept chilled for subsequent use. Ifappropriate, the homogenate was sonicated using 10-second bursts at maximum power from an Ultrasonic System Model 1000 Insonator. During this procedure care was taken to maintain a temperature below 10°C, and following this procedure the homogenate was returned to 0-5 °C. A determination of the homogenate's protein concentration was conducted, using the Bio-Rad protein assay kit, withbovine serum albumin as the standard. Absorbance was measured at 595 nm. Typical reaction tubes were prepared by the addition of 0.5 ml o 0.1 Mphosphate buffer, 0.5 ml (...truncated)