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I956 5. The countercurrent-distribution technique has also been used for studies of the action of aqueous HCI on protoporphyrin and its methyl ester. We thank Professor C. Rimington, F.R.S., for his continuous encouragement and support and Miss B. E. Tooth for technical assistance. We are grateful to B. Lewis and Co. for generous supplies of chicken blood. During part of the time that this work was carried out, one of us (J.E.F.) held a Foulerton Research Fellowship of the Royal Society. The Nuffield Unit is supported by a grant from the Nuffield Foundation to Professor C. Rimington.

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THE recent isolation of asparagine from an enzymic digest of edestin [Damodaran, 1932] provided the first direct evidence for the existence of a dicarboxylic acid amide in the protein molecule. As was pointed out in that paper it is, perhaps, from the point of view of demonstrating the validity of the amide hypothesis more important to isolate glutamine, because the majority of proteins on acid hydrolysis yield much larger amounts of glutamic acid than aspartic acid. But glutamine was known to be unstable in aqueous solution [Chibnall and Westall, 1932] so that at the end of the period required for the digestion of a protein to the amino-acid stage only a portion of the glutamine set free would still be present as such in the digest. Furthermore, the work of Schulze on the isolation of glutamine from plant extracts suggested that the separation of this amide from the digest would be difficult, and it was for these two reasons that the isolation of asparagine, which is stable in aqueous solution, and which crystallises readily in the presence of other amino-acids, was first attempted. With the experience thus gained a successful attempt has now been made to isolate glutamine from an enzymic digest of gliadin. This protein was chosen because of the high proportion of glutamic acid which it gives on acid hydrolysis; in terms of total protein-N it contains 25-7 % of amide-N, 22-9 % of glutamic acid-N, 1-17 % of hydroxyglutamic acid-N, and only 05 % of aspartic acid-N. In other words, if the validity of the peptide and amide hypotheses be assumed the gliadin molecule should yield 42-2 % of glutamine.At the outset an unforeseen difficulty arose, in that this prolamine was not as susceptible to the action of proteolytic enzymes as, for example, the edestin used in the asparagine research. While with this, and many other of the better known proteins, the successive action of pepsin, trypsin and either intestinal erepsin or yeast dipeptidase could accomplish the liberation of 90 % of the amino-N obtained on complete hydrolysis [Damodaran, 1932; Frankel, 1916], with gliadin it was found that the same enzymes split little more than 70 % of the total peptide bonds. The phenomenon, which invites further

INTRODUCTION. ONE of the most important corollaries of the peptide hypothesis of protein structure is that the dicarboxylic amino-acids exist in the protein molecule, partly or wholly, in the form of their amides. The correctness of this view, however, has never been directly demonstrated by the actual isolation of an acid amide from among the decomposition products of a protein. This is partly due to the fact that the method for the study of protein structure used, almost to the exclusion of all others, viz. that of acid hydrolysis, would break down any such acid amides into ammonia and the corresponding amino-acids. It was, in fact, through the observations of Nasse [1872-74] on the production of ammonia during the hydrolysis of proteins by acids and alkalis that the amide hypothesis originated. Fleurent [1893], who observed that large amounts of glutamic acid and ammonia were obtained by the hydrolysis of gliadin, was the first to suggest a possible connection between the ammonia and the dicarboxylic acids. These views attracted little attention till the development of the peptide hypothesis in 1902, when it was seen that they formed an excellent complement to the theories of Fischer and Hofmeister on the structure of protein, and they finally gained wide acceptance from the work of Osborne et al. [1906, 1908] who based their evidence on the apparent equality between the amount of ammonia and of total dicarboxylic acids that could be obtained from a large number of proteins. That most proteins on hydrolysis gave large amounts of glutamic acid and only very small amounts of aspartic acid, so that the amide-N might have its origin in the extremely labile glutamine (capable of being decomposed into ammonia and glutamic acid by boiling with water) and not in the more stable asparagine with which comparison was always made, was a possibility which seems to have been overlooked until Thierfelder and Cramm [1919] drew attention to it. These workers were however able to show that in synthetic dipeptides of glutamine (viz. glycyl-, alanyland leucylglutamine) the amide group was comparable in stability with that of asparagine, and further that the rate of ammonia liberation on acid hydrolysis was closely parallel with that from gliadin.