muramidase and ammonium-peroxydisulfate

Chymotrypsinogen was observed to have two bands in a low-pH gel electrophoresis system, though the protein was pure by other criteria. Other proteins have also been reported to give artifacts under these conditions. Removal of persulfate from the gel by pre-electrophoresis or by substituting riboflavin eliminated the artifacts. The affected amino acid residue was identified as tryptophan by titration of persulfate-treated proteins with 2-hydroxy-5-nitrobenzyl bromide and by the spectral method of Edelhoch. Persulfate-treated chymotrypsinogen had the same mobility as the artifact, while oxidation of Met-192 with hydrogen peroxide produced a protein with a different mobility.

Topics: Ammonium Sulfate; Animals; Cattle; Chymotrypsinogen; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Muramidase; Oxidation-Reduction; Trypsinogen; Tryptophan

A method was devised to quantitate N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) induced crosslinking of the ionic complex between [methyl-3H]thymidine labeled pBR322 DNA and lysozyme. This involved chromatography of the modified complex on Sephadex CM 25 with a high salt buffer that dissociated the non-covalently bound complex and permitted early elution of unbound [3H]DNA. The crosslinked complex was then eluted with buffer at pH 10.5. The amount of crosslinking was a function of the carcinogen concentration in the reaction mixture containing the complex. Addition of the spin trap nitrosobenzene or the radical trap 2,2-diphenyl-1-picrylhydrazyl to the complex prior to the addition of N-AcO-AAF decreased crosslinking. Reaction of the carcinogen with a solution of [methyl-3H]thymidine labeled Escherichia coli DNA led to the formation of tritiated water that was azeotropically distilled from the mixture. If nitrosobenzene was added prior to the carcinogen, the amount of tritium water released was depressed. Addition of N-AcO-AAF to a mixture of acrylamide and bisacrylamide increased its relative viscosity. Far u.v. irradiation of the complex or reacting it with ammonium persulfate that dissociates to the sulfate anion radical, SO(4), induced crosslinking. While these observations support a free radical mechanism for crosslinking, whether the free radicals arise from heterolytic or homolytic cleavage of N-AcO-AAF remains undetermined.

Topics: 2-Acetylaminofluorene; Acetoxyacetylaminofluorene; Ammonium Sulfate; Biphenyl Compounds; Chromatography, Ion Exchange; Cross-Linking Reagents; DNA; DNA, Bacterial; Escherichia coli; Free Radicals; Hydrazines; Muramidase; Nitrobenzenes; Picrates; Plasmids