Question

use TNBS to determine protein hydrolysate(peptide content)，but can not get results, please analysis what reasons can induce this ?

Answer 1

An accurate, reproducible and generally applicable procedure for determining the degree of hydrolysis of food protein hydrolysates has been developed. The protein hydrolysate is dissolved/dispersed in hot 1% sodium dodecyl sulfate to a concentration of 0.25-2.5 × 10-3 amino equivalents/L. A sample solution (0.250 mL) is mixed with 2.00 mL of 0.2125 M sodium phosphate buffer (pH 8.2) and 2.00 mL of 0.10% trinitrobenzenesulfonic acid, followed by incubation in the dark for 60 min at 50 °C. The reaction is quenched by adding 4.00 mL of 0.100 N HCl, and the absorbance is read at 340 nm. A 1.500 mM L-leucine solution is used as the standard. Transformation of the measured leucine amino equivalents to degree of hydrolysis is carried out by means of a standard curve for each particular protein substrate.