Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by crosslinkage

Publisher Website
Google Scholar
Abstract
Protease-free bovine pancreatic DNase (1.6 .times. 10-4 mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 .times. 10-2 mmol) at pH 10.5 with imidazole (2.4 .times. 10-2 mmol) as the catalyst in the presence of 4,4''-dithiodipyridine (4.2 .times. 10-2 mmol). The product obtained after 16 h at 4.degree. C, 2-acetamido-4-(4''-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 .+-. 0.13 mol of mixed disulfide/mol of DNase. RNase was thiolated in a similar manner, but under N2 in the absence of 4,4''-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 .+-. 0.11 mol of sulfhydryl groups/mol of RNase. The coupling of RNase to DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25.degree. C for 90 min. The hybrid enzyme (yield 25-33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase and homodimers by gel filtration, affinity chromatography and salting-out chromatography. The purified enzyme contained 1 molecule each of DNase and RNase and hydrolyzed thymus DNA and yeast or tRNA with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substate, phage f1 DNA .cntdot.[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the 2 enzymes offers the possibility in vivo of delivering 2 enzymes that differ in size, charge and biological function to the same site at the same time. [Since certain cancer viruses reproduce via the viral RNA genome and an RNA-dependent DNA polymerase, an enzyme capable of acting on the RNA and the DNA strands, both alone and in combination, is of interest in experimental cancer therapy.].
Keywords

This publication has 3 references indexed in Scilit: