On Determining the Chemical Structure of Proteins 275 



absence of tryptophan from the molecule were also strongly in its favor. 

 As a first step in the investigation, detailed amino acid analyses were 

 performed so that an accurate balance sheet in terms of amino acids 

 could be kept as the structural studies progressed. Although the quan- 

 titative amino acid analysis of a protein hydrolyzate no longer presents 

 serious problems, it has become apparent that the number of amino 

 acids that may decompose on hydrolysis and the extent of the decom- 

 position may vary from laboratory to laboratory and from protein 

 to protein. Possibly this variation could be minimized if the tempera- 

 ture at which the hydrolysis is conducted were precisely controlled. 

 Some decomposition during acid hydrolysis has been noted at one time 

 or another for serine, threonine, cystine, tyrosine, aspartic acid, glu- 

 tamic acid, proline, methionine, histidine, lysine, and arginine. 2 - 3 - 15 " 17 

 (Tryptophan, of course, decomposes nearly completely.) The variable 

 nature of this decomposition requires that individual corrections must 

 be worked out for each protein studied. Analysis after two times of 

 hydrolysis, say 20 and 70 hours, permits extrapolation to zero time and 

 seems to yield the most accurate results obtainable at present. The 

 longer time of hydrolysis also allows some estimate to be made of 

 those amino acids, such as valine or isoleucine, originally bound in 

 peptide linkages resistant to acid hydrolysis. The amino acid com- 

 position of ribonuclease given in Table 1 was determined in this 

 manner. 17 Corrections for decomposition had to be made for serine, 

 threonine, cystine, tyrosine, aspartic acid, glutamic acid, proline, and 

 arginine. Isoleucine was liberated slowly. Despite these corrections, 

 however, the number of residues of each amino acid present to the 

 extent of ten residues per mole or less is known with considerable 

 assurance. For those amino acids present in larger quantities, a rea- 

 sonable experimental error of ±4%, coupled with the uncertainties 

 introduced by corrections, may cause an error of ± one residue in the 

 final value. With proteins larger than ribonuclease, this limitation 

 might extend to most of the amino acids. 



End-group analyses of ribonuclease were performed by Anfinsen, 

 Redfield, Choate, Page, and Carroll. 18 When the DNP technique of 

 Sanger was employed, it was established that ribonuclease consisted 

 of a single peptide chain bearing lysine as the amino-terminal amino 

 acid, followed in turn by glutamic acid, threonine, and alanine. By 

 the use of carboxypeptidase, the carboxyl-terminal amino acid was 

 found to be valine, followed back along the peptide chain by phenyl- 

 alanine, isoleucine, or leucine, alanine, tyrosine, and methionine in un- 

 determined order. On the basis of this information and the analytical 



