Pancreatic Deoxyribonuclease

THE ROLE OF DIMERIZATION IN THE REVERSIBLE THERMAL INACTIVATION AT ACID pH

From the ¹ From the Laboratory of Molecular Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014

Crystallized preparations of pancreatic deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5) were further purified by gradient chromatography on diethylaminoethyl cellulose and phosphocellulose and used to study the reversible thermal inactivation in acidic media which was originally described by Kunitz. Changes in enzymatic activity and in a typical "tyrosine difference spectrum" suggest that the thermal transition is an interconversion between two monomeric states. Equilibrium and kinetic data for this process were collected over a limited temperature range. Circular dichroism and optical rotatory dispersion results indicate that the thermal transition reflects a relatively limited disruption of structure (compared to that occurring in concentrated solutions of guanidine·HCl or urea).

DNase which has undergone the thermal transition and then has been rapidly chilled exists largely as an enzymatically inactive dimer. The dimer was isolated by gel filtration; the apparent association constant is extremely large (K_assoc = 2 to 4 x 10⁷ liters per mole at 0°).

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