By this technique we have obtained milligram amounts of essentially pure enzyme and there appear no technical limitations to scaling up the process for the production of larger amounts. The uses and future of phosphocellulose columns in the purification of enzymes which act on nucleic acids have been discussed by Eley; however one difficulty arises in the phosphocellulose chromatography of the Lactobacillus enzyme.After elution of the enzyme with RNA from the first phosphocellulose column and throughout the second chromatography the presence of contaminating RNA interferes dramatically with the enzyme assay. In contrast to Eley's studies with chicken pancreas nuclease the RNA is not removed completely on the second (salt elution) column. We have resorted to extended incubation periods allowing degradation of the accompanying RNA followed by dialysis to eliminate the RNA. Unfortunately contaminating RNA (<2% by weight) is often still retained. However, when fraction IV or V enzymes are subjected to electrophoresis on acrylamide gels the enzyme which can be recovered from the gels is now free of detectable RNA. We anticipate that the use of preparative disc gel electrophoresis will provide us with completely pure protein for our future studies.
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