1. Native, lyophilized and urea denatured hemoglobins were carboxymethylated with bromoacetic acid in the presence of MgO. The optimum pH of the reaction to attack histidine residues in hemoglobin was pH 9, and tyrosine reacted only slightly at this condition. The reaction ended within 10 hours at 30° and about 60 per cent of the total histidines reacted.
2. After treatment with 2 to 8M urea, additional 7 to 22 per cent of the total histidine residues reacted with bromoacetic acid in comparison to those with native one, whereas the lyophilized hemoglobin reacted in the same way as the native.
3. The absorption spectrum at the Soret region was remarkably diminished by the carboxymethylation and particularly a big change was observed with the hemoglobin denatured with 2M urea.
The author wishes to express gratitude to Prof. Y. Oshima and Prof. M. Funatsu for their invaluable advice and criticism during this work, and to Mr. S. Abe for assistance in the preparation of hemoglobin.