I should like to express my thanks to the following: to F. C. Bawden, under whose auspices the work was carried out, and N. W. Pirie, for advice; to P. H. Gregory for potato samples and help in planning the experiments; to H. M. Sinclair for a culture of Phycomyces blake8leeanus; to my brother, A. P. Meiklkjohn, for calling my attention to the possibilities of the method, and to E. M. Crook and D. J. Higgons for supplies of solanin6 and solanidine.

ALTHOUGH glutamine has been known for many years to be a constituent of certain plant tissues [Schulze and Bosshard, 1883], no method whereby this substance can be separately distinguished from asparagine and determined with accuracy has appeared until recently. Glutamine has indeed frequently been isolated but its properties are such that isolation has only qualitative significance. Chibnall and Westall [1932] noted that the amide group ofglutamine is extensively hydrolysed by heating at 1000 for 3 hours at PH 8, whereas asparagine is scarcely affected by this treatment. Accordingly they suggested that the glutamine

The carboxyl groups of a protein can be esterified with diazomethane, but more conveniently with methanolic hydrochloric acid. In the present research, however, neither reagent has proved to be entirely satisfactory, for in our hands the first has not given full esterification and the second would do this only at the expense of the amide N, which we were particularly anxious to keep intact. Fraenkel-Conrat & Olcott (1945) were the first to show that the concentration of mineral acid (0-02 to 01 N) required to catalyse the reaction with methanol was very much less than previous workers had employed for the purpose. They claimed that 97 % of the carboxyl groups of polyglutamic acid from Bacillu8 8ubtili8 could be esterifled by treatment with methanolic 0-05N-HCI at 22-24°for 24 hr. and that several proteins, including insulin, were fully esterified under similar conditions in the presence of 01IN-HCI. They mentioned that there was no loss of amide N under such treatment, though data in support of the assertion were not presented. Mommaerts & Neurath (1950) repeated the experiments with insulin, and confirmed that full esterification was apparently achieved with methanolic 0-1 N-HCI at 250 in 24 hr. They claimed that the ammonia liberated under these conditions amounted to not more than 2 % of the amide N.Our own observations do not support the contention that 0 1 N-HCI can be used as a catalyst in the esterification of proteins in this way without loss of amide groups. With insulin the ammonia produced in 24 hr. at 25°represents about 6-6 % of the amide N, a value much greater than the abovementioned workers seem to have suspected. Lowering the concentration of hydrochloric acid spares the amide N but unfortunately full esterification is not then achieved. We have thus been obliged to prepare the ester with methanolic 0 1 N-HCI, knowing that when this is used subsequently to determine inter alia the amide distribution of the protein, we had already set a limit to the accuracy with which this could be done. Another interesting side-reaction of protein esterification has been traced to N-O acyl migration in serine and particularly threonine residues. EXPERIMENTAL MateriakProtein samples, and methods for determining N, ammonia N and amide N were described in the preceding paper (Chibnall, Mangan & Rees, 1958b). Methoxyl. The Zeisel procedure of Pregl (1937) was used. Estimations on 20-30 mg. samples agreed to within 2%.Methanol. This was boiled under reflux for some hours with Mg turnings and then distilled.Methanolic hydrochloric acid. An approx. 2N-solution was prepared with dry HCI gas and stored at -150. As the acidity falls on keeping it is necessary to titrate a sample immediately before use. MethodsEstimation of the extent of esteriftcation. The extent of esterification of the total free carboxyl groups was followed by determination of methoxyl. As ,-lactoglobulin contains methionine, which yields methyl iodide on treatment with HI, the (apparent) methoxyl content of the protein itself was subtracted from that...

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