An Ultraviolet Spectrophotometric Micromethod for Studying Protein Hydrolysis

A spectrophotometric method for the determination of amino acids and peptides based on measurement (620 mp) of the blue color formed when their copper salts were converted to the copper salt of alanine has been described (1).Incidentally, it was observed that the copper salts of amino acids have absorption maxima near 235 rnp.Ley and Hegge (2) showed that the copper salts of glycine and alanine have absorption maxima near this wave-length.The molar absorbancy index' of the copper salt of alanine was 56 at 620 rnp and approximately 6200 at 235 m/l; therefore, measurements at 235 rnp were over 100 times as sensitive as those at 620 w.The present paper describes a micromethod for the estimation of amino acids based on the ultraviolet absorption of their copper salts, with particular regard to use of the method in studying the rate of protein hydrolysis. EXPERIMENTALA Beckman quartz spectrophotometer, model DU, with 10 mm.quartz cuvettes was used.Amino acids, peptides, and proteins were the same as those described previously (1).Copper Chloride-8.52 gm. of reagent grade cupric chloride dihydrate per liter of water solution (0.05 M).Sodium Borate Buj'er, pH 9.1 to 9.2-40.3gm. of reagent grade, anhydrous sodium tetraborate in 4 liters of water sdlution; filtered.For use in the procedure, 6.0 gm. of reagent grade sodium chloride were dissolved in 100 ml. of borate solution (4) ; this sodium chloride-containing solution will be referred to as buffer solution.Method of Analysis-To 5.0 ml. of buffer solution in a 25 ml.glassstoppered Erlenmeyer flask were added 5.0 ml. of a water solution of an appropriate concentration of the test substance.0.1 ml. of copper chloride solution was then added, and the flask was shaken and let stand for 10 minutes at a convenient room temperature, the same temperature (f 1") being used for all determinations.The suspension was centrifuged in a 1 Defined by Gibson (3).65