Direct Measurement of Enzyme Activity with Infrared Spectroscopy

Abstract

A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca2+-ATPase: The substrate concentration can be followed using the infrared absorption of the α- and β-PO2−phosphate groups of ATP, and the product concentration can be followed using the P032- absorption of Pi and of the fl-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly—that is, no activity assay that converts the progress of the reaction into an observable quantity is required.