Parameters of biological freezing damage in simple solutions: Catalase: II. Demonstration of an optimum-recovery cooling-rate curve in a membraneless system

Abstract

Seeded solutions of catalase in neutral 10 m M potassium phosphate buffer exhibited characteristic rate dependencies for freeze-thaw damage: Damage increased as the cooling rate was increased, and as the warming rate was decreased. The pattern of warming-rate dependence was independent of the prior cooling rate and also of the addition of KCl or of NaCl to the buffer. In contrast, the cooling-rate curve became almost flat upon addition of 0.1 M KCl, suggesting increased damage from concentrating solute at low cooling rates. In the presence of added NaCl, frank optimum-recovery cooling-rate curves were generated. At low NaCl levels (less than 10 m M) the optimum occurred at 0.5 °C/ min; at 27 and 81 m M NaCl, the optimum shifted to 5 and 20 °C/min, respectively. By comparison with KCl, it appears that the major factor causing damage at low cooling rates in NaCl is acidification. The factor causing damage at high cooling rates remains obscure. The argument that it is due to the trapping of the enzyme molecules at interfaces at high dilution, to be subsequently damaged by shearing stress or dehydration during the recrystallization attending slow warming, is mitigated by the finding that inactivation remains a function of the initial enzyme concentration at all cooling rates. The possibility that a particular conformational state is trapped in an unfavorable temperature zone was also considered: Three simple models were formulated, and the relative order of recovery was deduced for the possible sequences of fast and slow cooling and warming. The permutation observed for catalase was inconsistent with any of these three mechanisms, although they may be pertinent for the red cell and other systems. A final possibility, not yet explored, is that rapid cooling causes damage by producing nonequilibrium freezing, with large deviations of pH and/or solute concentration from those expected at equilibrium.