PP SAREE SLAOAa tae eee Popp/Batuirt /tlinscH/CONRAD 212 leucine. Iscleucine-"H radioactivity counts were also used to calculate the percentage of isolcucine eluting from the preparative column which was actually pooled for the quantitative analysis of isoleucine in the protein hydrolysate; i.c. the small quantity of isoleucine that did not completely resolve from leucine could be included in computing the quantity of isoleucine in the original sample. The pooled eluate must not contain much leucine becausc it would interfere with an accurate determination of the small quantity of isoleucine there. After adjusting the pH to 2.2 by adding 1 N HCl, the quantity of isoleucine in the pooled eluate was determined using a Beck- man Model 120C aminoacid analyzer [11]. The frequency at which isoleucine substitutes for other amino acids in human hemoglobin was calculattd by dividing the nanomoles of isoleucine by the nanomoles ofall other amino acids in the sample. Results About 3%of the total protein, mostly molecules of higher molecular weight, were separated by molecular seiving over Sephadex G-200. Based on an average isoleucine content of 5% for nonhemoglobin proteins, the carbon monoxyhemoglobin fraction still contained 3-4% nonhemoglobin protein after molccular seiving. This was removed by repeated chromatography of chemically derivatized hemoglobin over carboxymethylcellulose. The substitution frequencies of isoleucine for other amino acids in the highly purified hemoglobin from 15 controls of ages 20-60 years are shown in table I. Data on 13 Marshallese who were exposed to fallout radiation in 1954 are prescnted in table I to indicate that the method does detect individuals who have hemoglobin with a higher isoleucine content, presumably due to radiation-induced base substitutions in erythropoietic stem cells. Oneof the controls, sample 1547, showed a significantly higher value than the rest and if was not included in making the following calculations. The averageisolettcine substitution frequency for the 3 laboratory personnel and the 12 Marshallese controls is 2.99 x 10-*, and the computed line of best fit has a positive slope of 0.0296 x 10-*/year. The isolcucine substitution frequency in the separated a- and §-chains of a few samples was determined to show that the values equalled those obtained for the plobin, although the substitution frequency in the B-chain was higher than in the a-chain (table I),