Publication Date

5-1993

Advisor(s) - Committee Chair

Valgene Dunham, Claire Rinehart, Frank Toman

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

A deoxyribonuclease (DNase) has been isolated from a purified preparation of soybean nuclei. The procedure, involving solubilization of proteins using ammonium sulfate, Sephadex G-75, DEAE-Cellulose and CMSephadex chromatography and non-denaturing polyacrylamide gel electrophoresis, resulted in a 554-fold purification with a yield of 6% of the enzyme. The purified enzyme had a molecular weight of 60 KDa as monitored by non-denaturing polyacrylamide gel electrophoresis. In addition, high-molecular-weight (HMW) soybean DNA, essentially free from protein, RNA and other contaminates, has been isolated from soybean to be used as a substrate for the enzyme. Enzyme activity was directly proportional to the concentration of enzyme and substrate as determined by a spectrophotometric assay. In the presence of divalent cations (Mn2+), the enzyme hydrolyzed soybean DNA, calf thymus DNA and pBR322 DNA but was inactive with yeast RNA. The enzyme was partially inhibited by KC1. Endonuclease activity was indicated on agarose gels using covalently closed circular pBR322 DNA and spectrophotometric analysis of assay products following Sephadex G-10 chromatography. The enzyme had a Km for soybean DNA of 4.5 μg/ml and a Vmax of 0.02 ΔA260/min.

Disciplines

Biology | Life Sciences

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