In the course of an investigation on the reaction between biogenic amines and proteolipids (1)) methods were required to analyze for proteins and amines in mixture. The method of Satake et al. ( 2) proved to have some easily corrected disadvantages and some unexploited potentialities which make it ideal for the investigation in progress. The development of the methods reported herein was made possible by the observations that the absorption spectrum of the product of the reaction between 2,4,6-trinitrobenzenesulfonic acid1 and derived amines is not identical to the absorption spectrum of the corresponding purified TNP derivative and that the TNP derivatives had useful solubility properties. EXPERIMENTAL Materials. TNBS was the Eastman product, recrystallized from 1 A.? HCl (3). The organic solvents were C.P. chemicals, redistilled before use. The amines and amino acids were Calbiochem products, grade A. Less pure peptides and unstable amines were repurified by formic acid gradient elution from columns of Amberlite IRC-50. a-Casein was prepared from food-grade casein by the method of Warner (4). Other proteins were crystalline products from Sigma.
Procedure A: Solutions of amines, amino acids, OT proteins. To the sample (less than 0.2 pmole of amine or amino acid and less than 0.4 mg of protein) in 1.0 ml of 0.10 M K,B,O,, add 0.10 ml 0.03 M TNBS. Mix and allow to react for 25 min at 2426" or 10 min at 40", whichever is more convenient. Quench the arylation reaction by the addition of 2.0 ml of cold (0") methanol. Read the absorbance of amines and nonaromatic amino acids at 420 rn+u, and of proteins and aromatic amino acids at 340 rnp. Compare readings with standards similarly treated.