Labelling of cysteine proteinases in purified lysosomes.

NADH could not be freeze-stored at -25°C in 50 mMsodium phosphate buffer (pH range 6-8 20°C) without serious loss of the nucleotide; however, no losses were seen upon freeze storage in 50 mM-Hepes buffer (pH range 7-8, 20°C). The rate of NADH degradation was greater (approx. 2-fold) in 50 mMthan in 5 mM-sodium phosphate buffer, although NADH values at both buffer concentrations were similar (5% remaining) after the 2 day freezing period. However, NADH stored in liquid nitrogen in 50 rnM-sodium phosphate buffer, pH 7.0, retained XY‘% of its pre-freeze value over a 2 day storage period. The rate of freeze-induced NADH degradation was not affected by the number of times the sample was thawed and refrozen. Very little difference was seen in the degradation rate of single freeze samples over that of the multifreeze sample. Thus, it would appear that time spen! in the frozen state is the major factor responsible for NADH degradation other than the freezing process itself. NADH incubated at 4°C at pH 3.7, 5.0, 6.0, 7.0, 7.5 and 8.0 showed considerable degradation at acidic pH. The degradation profile at pH 3.7 at 4°C was very similar ( I , , , 5 h ) to that seen with 50 mM-sodium phosphate buffer pH Bovine serum albumin retarded the freeze-induced pH changes in phosphate buffers with higher protein concentrations being more effective (7.5 mg of BSA/ml prevented any pH change). Similarly, dimethyl sulphoxide and glycerol retarded the pH changes with 30% of either cryoprotectant preventing any pH change. On freezing yeast alcohol dehydrogenase and pig liver cytosolic aldehyde dehydrogenase in sodium phosphate and Hepes buffers, pH 7.0, in both cases enzyme losses are much greater in sodium phosphate buffer than Hepes buffer and were greater at -90°C than at 196°C. Sample activity retention showed little dependence o n the freezing rate as samples frozen rapidly by immersion in liquid nitrogen and then transferred t o -25°C showed similar losses t o those frozen slowly from room temperature t o 25°C. Protein freeze storage has. in the past, been more o f an art than a science and any rules governing such freezc storage, if indeed any exist, have been rather empirical. The major cause of protein denaturation upon freezing could be due t o the development of an acidic pH. although the extent of such denaturation is variable with diffcrent proteins (4 1. It is suggested that the following steps, if followed progressively, might help to reduce the time spent and the material lost in the usually ‘hit and miss art’ o f protein freeLe storage: ( 1 ) Where possible, store samples in zwitterionic buffers which show little pH change on freezing (such as Hepes). (2 ) Storage at liquid nitrogen temperatures. (3) If storage in inorganic buffers cannot be avoided then: ( u ) determine the pH change of buffer upon freezing by the indicator method; ( h ) replace one buffer, where possible, with another in which the pH change is smaller (such as potassium phosphate for sodium phosphate); (c) freeze at high protein concentration to prevent pH changes, the indicator method can be used t o determine the minimum protein concentration required; ( d ) use small volumes to allow for rapid freezing and thawing; and ( e ) use additives: test the cryoprotectant‘s ability to prevent buffer pH changes by the indicator method; the cryoprotectant action might be protein specific and require a lengthy test procedure.