The Nature of the Random Experimental Error Encountered when the Kinetics of Acetylcholine Hydrolase are Determined

does not fully deplete the enzyme of endogenous nucleotides. We agree with Garrett & Penefsky (1975) that the ATPase activity is constant for long periods (up to 3 weeks) when the enzyme is stored in 50% glycerol even in the absence of ATP. After treatment of ATPase by the Garrett & Penefsky (1975) procedure, reaction of the enzyme (in 50 % glycero~/lOOm~-Tris/H,SO,, pH 8.0) with 4-chloro-7-nitrobenzofurazan still led to inactivation of the enzyme after modification of one tyrosine group per enzyme molecule. This observation is of interest since our previous work on this reaction was done in the presence of 4 m ~ added ATP. Clearly the added ATP that was necessary to stabilize the enzyme (Ferguson et al., 1975a) does not cause the asymmetry of the enzyme towards 4-chloro-7-nitrobenzofurazan. The absorption spectrum of the ATPase in 50% glycerol at pH8.O showed an unexpectedly strong which was absent when the ATPase was dissolved at pH8.0 in a buffer lacking glycerol. A similar increase in A2,,, was observed when lysozyme and bovine serum albumin were transferred into buffers that contained 50 % glycerol. These effects were not seen at pH7.5. The absorbance at 240nm can be attributed to ionized phenolic tyrosine groups, as N-acetyltyrosine ethyl ester showed very similar behaviour. Glycerol therefore seems to increase the ionization of the phenolic groups of tyrosines.