On Determining the Chemical Structure of Proteins 279 



that would not react elsewhere in the molecule. One of the most 

 specific reactants for an — SH group is another — SH group in the 

 presence of oxygen. Whether protein— SH groups fonned by reduction 

 could be induced to react completely in the presence of oxygen with 

 an excess of another SH compound such as cysteine or thioglycolic acid 

 remains to be determined. Under any circumstances, the question 

 of how to deal with disulfide bridges * in proteins containing trypto- 

 phan may prove to be one of the crucial bottlenecks in future work 

 with protein structure. 



Once a single peptide chain free from cross bridges is obtained, 

 three general approaches to the determination of its structure are avail- 

 able. In one, the sequence of amino acids is derived by hydrolyzing 

 the chain into relatively small fragments employing more or less 

 random hydrolysis with strong acid. The large number of di-, tri-, 

 and tetrapeptides fonned must then be separated and the structure 

 of each determined. This is essentially the approach utilized by 

 Sanger, but, despite his notable success, this method has serious draw- 

 backs which he well appreciated. 32 An enormous number of peptides 

 are formed after such treatment, most of them in relatively poor yield. 

 The problems of fractionating and isolating pure peptides from such 

 a mixture are great. Although each individual peptide is of relatively 

 simple structure, this advantage is offset by the large number of 

 peptides requiring study. It is doubtful whether such an investigation 

 could be completed successfully for peptide chains much longer than 

 the thirty amino acids contained in the B chain of insulin. It does 

 not appear to be a generally feasible approach for determining the 

 structure of a large number of complex proteins. 



A second possible method of determining the sequence of amino 

 acids in a peptide chain is by stepwise degradation starting at either 

 the carboxyl or amino end. Promising techniques for accomplishing 

 this are available, or on the horizon, and it may well be the approach 

 of choice for small peptides. It seems doubtful, however, whether 

 any stepwise procedure will be suitable for the degradation of long 

 chains of amino acids such as occur in intact proteins. The losses 

 involved in each step seem insupportable. If, for example, 1 gm. of 

 a protein were degraded stepwise and a yield of 75% obtained at each 



* The existence of diester phosphate bridges in proteins, and methods for cleav- 

 ing them, have been suggested by the work of Perlmann. 33 Diester sulfate bridges 

 are also a possibility after the finding of Bettelheim 34 that tyrosine-O-sulfate 

 is a constituent of fibrinogen. 



