A spectrophotometric method for the measurement of ribonuclease activity.

Digestion of yeast nucleic acid by ribonuclease is accompanied by a shift in the ultraviolet absorption spectrum of the substrate towards the shorter wave-lengths (Fig. 1). The shift is not due to the liberation of free acid which takes place during digestion (1, 2), since the effect of increasing the hydrogen ion concentration is to shift the absorption spectrum of yeast nucleic acid in a direction opposite to that caused by ribonuclease; namely, towards the longer wavelengths (Figs. 2 and 3). The shift in the ultraviolet spectrum caused by ribonuclease is most distinct in the region of 290 to 305 ml.c, where the grad& decrease in the extinction (density) E = log IO/I during the initial stages of the digestion process can be readily measured. The determination of the rate of change in the absorption of the ultraviolet light can thus serve as a convenient method for measuring the concentration (activity) of the ribonuclease in the digestion mixture. The effect of varying the concentration of ribonuclease on the value of E at 300 rnp is shown graphically in Fig. 4, A. The value of E decreases linearly with time, at least during the initial phase of the reaction, the rate of decrease being nearly proportional to the concentration of enzyme in solution, as shown by the values for the slopes of the lines. Hence