Determination of free amino groups in proteins by trinitrobenzenesulfonic acid

A sensitive method to determine the free amino groups in proteins is presented that makes use of the reagent 2,4,6-trinitrobenzenesulfonic acid. The relationship between absorbance and concentration (up to 1 mg/ml) was linear and the color was developed under more gentle conditions than with the ninhydrin method. It was found that sodium dodecyl sulfate was bound to some of the ϵ-amino groups of lysine in bovine serum albumin, ovalbumin, and human γ-globulin and rendered the involved amino groups unreactive toward TNBS. Partial reaction of 4 M potassium thiocyanate at 40°C for 2 hr with the free amino groups in bovine serum albumin, ovalbumin, human γ-globulin, and lactalysate was demonstrated by this method. The method was useful to study the reaction of the free amino groups of bovine serum albumin with formaldehyde.