A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm ฯช1 ) and diphosphate (1085 cm ฯช1 ) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPaseactive RNA-packaging enzyme (protein P4) of bacteriophage 6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 ฯฎ 1.3 mol โ
mg ฯช1 โ
min ฯช1 at 40ยฐC) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (ฯณ 10 ฯช6 g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.