A simplified purification procedure for mung bean nuclease has been developed yielding a stable enzyme that is homogeneous in regards to shape and size. The nuclease is a glycoprotein consisting of 29% carbohydrate by weight. It has a molecular weight of 39 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme contains 1 sulfhydryl group and 3 disulfide bonds per molecule. It has a high content (12.6 mol %) of aromatic residues. Approximately 70% of the enzyme molecules contain a peptide bond cleavage at a single region in the protein. The two polypeptides, 25 000 and 15 000 daltons, are covalently linked by a disulfide bond(s). Both the cleaved and intact forms of the enzyme are equally active in the hydrolysis of the phosphate ester linkages in either DNA, RNA, or adenosine 3'-monophophate. The enzymatic activity of mung bean nuclease can be stabilized at pH 5 in the presence of 0.1 mM zinc acetate, 1.0 mM cysteine, and 0.001% Triton X-100. The enzyme can be inactivated and reactivated by the removal and readdition of Zn2+ or sulfhydryl compounds.

Under conditions which favor the duplex structure of DNA, mung bean nuclease catalyzes a limited number of double-strand cleavages (probably less than 50) in the interior of native T7 DNA. However, under conditions which are not as favorable to a tight helical structure, the large duplex polymers previously produced are completely degraded from their termini with a continuous accumulation of mono-, di-, and trinucleotides. The terminally directed activity is an intrinsic property of the enzyme molecule because (1) it is inactivated and reactivated in parallel with the single-strand activity and (2) the two activities coelectrophorese on analytical gels. Kinetic measurements indicate that the apparent Km for the terminally directed hydrolysis of native DNA is relatively high. The pH optimum for both the hydrolysis of denatured DNA and the terminally directed hydrolysis of native DNA becomes more acidic with increasing salt concentration. The relative preference for single-stranded structures increases as the pH becomes more basic.

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