Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM.
An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised. The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM. Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP. The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed. This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured. It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.
Florini
J R
JR
Biology Department, Syracuse University, New York 13244.
D000818:Animals; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000032:analysis*; D008832:Microchemistry; D009243:NAD / Q000378:metabolism / Q000031:analogs & derivatives; D011817:Rabbits; D013053:Spectrophotometry
D009243:NAD; C003451:thionicotinamide adenine dinucleotide; D003402:Creatine Kinase