MEASUREMENT OF NANOGRAM QUANTITIES OF PROTEIN BY HYDROLYSIS FOLLOWED BY REACTION WITH ORTHOPHTHALALDEHYDE OR DETERMINATION OF GLUTAMATE

Two methods are described for determining protein by measurement of amino acids released by acid or base hydrolysis. One achieves sufficient sensitivity to measure as little as 3 ng of protein by reaction of the amino acids with orthophthalaldehyde and mercaptoethanol in a small volume, followed by dilution in 0.5 N NaOH for fluorometric measurement of the product. The other method is somewhat more sensitive. Released L-glutamate is determined enzymatically by reaction with glutamic dehydrogenase (EC 1.4.1.2) and NAD+; the NADH produced is amplified by enzymatic cycling. To avoid the use of sealed tubes and minimize dangers of contamination, hydrolysis is carried out in small droplets in oil wells. [Bovine serum albumin was used].