The implications for practice are discussed and recommendations for further research are made.

In order to study critically some physical characteristics and biological properties of insulin we needed an accurate measurement of the molarity of insulin solutions. Herskovits (1965) has measured the concentration ofinsulin by nitrogen analysis and published a value for the molar extinction coefficient of 5-96 x 103 at 277nm. This contrasts with the value of 5*22 x 103 obtained by Praissman & Rupley (1968) from dry-weight determinations corrected for counterion content. It occurred to us that this considerable difference could arise if the wrong value for the nitrogen content of insulin was assumed. The amide nitrogen atoms of insulin are known to be labile (Sundby, 1962) and commercial preparations ofinsulin have a degree ofheterogeneity that has been attributed to random loss of amide nitrogen (Carpenter & Chrambach, 1962; Mirsky & Kawamura, 1966). In an extreme case insulin could lose 6 amide nitrogen atoms/mol., thus decreasing its nitrogen content from 65 to 59 atoms/mol. (Sanger, 1960). Another potential source of error is the small but measurable amount of proinsulin that commercial preparations of insulin are known to contain (Schmidt & Arens, 1968). We have evidence that the most highly purified samples of insulin available from different commercial sources vary sufficiently in their amide nitrogen content to account for the high extinction coefficient obtained by Herskovits (1965), and we have established that the molar extinction coefficient for insulin is 5-53 x 103 at 277-5nm. We have also measured the molar extinction coefficient oftyrosine and find it to be 1-38 x 103 at 274-1nm. Ten-times-recrystallized bovine and porcine insulin from Novo Industri A.S (Copenhagen, Denmark), two different samples of six-timesrecrystallized bovine insulin from Boots Pure Drug Co. (Nottingham) and crystalline bovine insulin from Burroughs Wellcome Ltd. (Beckenham, Kent) were all generously supplied by the Companies concerned. L-Tyrosine was purchased from Sigma Chemical Co. (St Louis, Mo., U.S.A.) A.R. (NH4)2SO4 was dried and stored over P205 in an evacuated desiccator. Standard solutions of known nitrogen concentrations were made up by accurately weighing and diluting this dried salt.

Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The resultant preparations were fragmented in various ways and the ratio of tyrosine to monoiodotyrosine and di-iodotyrosine was determined in each fragment. This has allowed the distribution of iodine between the combined A-chain tyrosine residues and the individual B-chain tyrosine residues to be determined. The hormonal activity of each of these iodinated insulin preparations was measured from their effect on the production of (14)CO(2) from [1-(14)C]glucose by isolated adipose cells. The results were interpreted as meaning that the iodination of tyrosine residue A19 or B16 leads to the inactivation of insulin. Speculations are made about the nature of an interaction between insulin and a receptor site on the target tissue.

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