Intracellular alpha-keto acid quantification by fluorescence-HPLC
The methods for isolating the amino-acids yielded by hydrolyzed proteins, as introduced by Kossel and Fischer, and further developed by other workers, have given us most of our present knowledge of the composition of proteins, and still constitute the means of attaining the most complete information concerning their chemical nature. These methods are still encumbered by two inherent difficulties, however, which limit their application in problems involving the chemical nature of proteins. (1) A large supply of material is required, 20 or 30 grams of protein for the determination of the bases by Kossel’s method, while at least 100 grams, and preferably 300 or 400 grams, are used for the esterification. (2) The methods for determining most of the amino-acids are not quantitative. As a consequence, the most careful work leaves from a third to a half of the protein molecule still unaccounted for. The analysis outlined below’ is designed to enable one to attain an insight into the composition of proteins by methods which require but small amounts of material and yield approximately quantitative results, indicating the nature of all the nitrogenous products yielded by complete acid hydrolysis. The analysis is based, not on the isolation of the amino-acids, but on determinations of their characteristic chemical groups.