Raman spectral studies of nucleic acids series. part LXIX - A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphat e phosphohydrolase (NTPase) activity. The method relies upon simultaneous m easurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm-(1)) moieties o f the NTPase substrate and product, respectively. The reliability of the me thod is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4 ) of bacteriophage phi 6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 mu mol mg(-1) min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatilit y of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosph ates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advanta ges of the present protocol include conservative sample requirements (simil ar to 10(-6) g enzyme/protocol) and relative ease of data collection and an alysis. The latter conveniences are particularly advantageous for the measu rement of activation energies of phosphohydrolase activity. (C) 1999 John W iley & Sons, Inc.