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

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Abstract
A [chicken] inducer protein, the vegetalizing factor, which causes amphibian ectoderm to differentiate into endoderm was labeled with carrier-free 125I(2000 Ci/m-atom) to specific activities of 17-32.mu.Ci/.mu.g 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 labeled to specific activities of 0.8-2.3 .mu.Ci/.mu.g 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 considerably reduced, when under more stringent conditions, 60-70% of the amino groups were methylated. NMR spectroscopy showed .apprx. 1/3 of protein amino groups are dimethylated under these conditions. The factor could be labeled with N-(propionyloxy)succinimide without a loss of the biological activity, when very few amino groups were substituted. The factor can be labeled to high specific activity without loss of its inducing activity by reductive methylation, which is therfore, the most suitable method.

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