The measurement of amino groups in proteins and peptides

Abstract

A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5–200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30–50 times faster than with ∈-amino groups of model compounds. The ∈420 of a trinitrophenylated thiol group was found to be 2250m-1·cm-1. The reaction with several amino acids, peptides and proteins is presented. The ∈420 of a typical α-amino group was found to be 22000m-1·cm-1 and that of an ∈-amino group, 19200m-1·cm-1. Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.