Radioiodination with125I and Reductive Methylation with Tritium of a Vegetalizing Inducer Protein. Specific Radio-Activities and Effect on Biological Activity

Abstract

An inducer protein, the vegetalizing factor, which causes amphibian ectoderm to differentiate into endoderm was labelled with carrier-free 125I (2000 Ci/m-Atom) to specific activities of 17-32 micronCi/microgram protein. The partial loss of the biological inducing activity is not caused by the iodination of the factor but is due to the oxidation and reduction steps in the iodination procedure. By reductive methylation with formaldehyde and [3H]NaBH4 under mild conditions the factor was labelled to specific activities of 0.8-2.3 micronCi/microgram protein depending on the specific activity of the [3H]NaBH4 (5-15 Ci/mmol). Under these conditions up to 25% of the amino groups of the protein were methylated without a reduction of the biological activity of the factor. The inducing activity of the protein was however considerably reduced, when under more stringent conditions 60-70% of the amino groups were methylated. It was shown by NMR spectroscopy that about one third of protein amino groups is dimethylated under these conditions. The factor could be labelled with N-(propionyloxy)succinimide without a loss of the biological activity, when very few amino groups were substituted. The experiments show that the factor can be labelled to high specific activity without loss of its inducing activity by reductive methylation, which is therefore the most suitable method.