The Complete Enzymic Hydrolysis of Proteins

The strong acids that are required for complete hydrolysis of proteins destroy some amino acids, such as tryptophan, asparagine, and glutamine. Other amino acids are destroyed to a lesser extent or in certain cases are released incompletely from peptide linkage. It is evident that these difficulties, as well as others, could be overcome by employing proteolytic enzymes as the sole agents for hydrolysis. The complete enzymic degradation of proteins was studied by several investigators in the late nineteenth and early twentieth century. Meissner (2), Kiihne and Chittenden (3, 4), Chittenden and Goodwin (5), and Neumeister (6, 7) demonstrated that pepsin and trypsin hydrolyzed proteins to produce a complex mixture of peptides called proteoses and peptones. These were considered to be the ultimate end products of proteolysis until 1901, when Cohnheim (8) showed that intestinal extracts (erepsin) hydrolyzed peptones to amino acids. Subsequent studies, in particular those of Frankel (9), showed that the combined action of pepsin, trypsin, and erepsin resulted in cleavage of over 90% of the peptide bonds in several purified proteins.1 Although it was evident from these early investigations that proteins could be degraded by enzymes to amino acids, later investigators did not develop a system for complete enzymic hydrolysis. This can be attributed to a variety of causes, but two major problems which only recently have been satisfactorily solved presented the most difficulty. Micromethods for the precise analysis and characterization of amino acids and peptides were not available, and secondly, purified proteolytic enzymes were not characterized sufficiently to be used as hydrolytic agents. With these considerations, we have developed methods that employ proteolytic enzymes for hydrolysis of proteins. Our studies demonstrate that digestion of a protein by papain (12), followed by treatment with the purified kidney peptidases, leucine aminopeptidase (13), and prolidase (14) results in essentially complete hydrolysis of all peptide bonds and gives high yields of tryptophan, glutamine, and asparagine.