DEOXYRIBONUCLEASE-I FROM RAT URINE - AFFINITY PURIFICATION, CHARACTERIZATION, AND IMMUNOCHEMICAL STUDIES

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000- fold to apparent homogeneity with a 14% yield by affinity chromatograp hy utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases . Isoelectric focusing electrophoresis revealed that all six isoelectr ic forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-ter minal amino acid sequencing were performed on the purified DNase I. Th e N-terminal sequence up to the 15th residue of the enzyme was identic al to that of rat parotid DNase I. The enzyme was found to be a glycop rotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule, The isoelectric mul tiplicity of the enzyme was partly due to differences in the sialic ac id content of the isoforms, Gel filtration on Superose 12 and electrop horesis on sodium dodecyl sulfate polyacrylamide gels indicated an app roximate molecular mass for DNase I of 32 kDa. The enzyme had an optim um pH of 6.5 and required divalent cations such as Ca2+ for its activi ty, Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin, An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the act ivity of the purified enzyme.