The determination of the substitution achieved at the alpha-amino, epsilon-amino and imidazole groups of proteins with special reference to derivatives of gelatin.

1. The diminution of the quantity of pigment formed in the ninhydrin colorimetric reaction or the titre in two types of formol titration of a protein after substitution is used to determine the degree of substitution achieved at the various amino groups of a protein. 2. The determination of the substitution achieved at alpha-amino plus in-amino groups by the ninhydrin colorimetric method as described by Cobbett, Gibbs & Leach (1964) has been modified to correct for the hydrolysis of the peptide chain which takes place during colour development. 3. The degree of substitution at the in-amino groups was determined by the formol titration carried out at pH9, essentially as described by the above authors. 4. Substitution at the alpha-amino plus in-amino plus imidazole groups was determined from the diminution of the titre of a modified formol titration. This titration was carried out by adjusting the protein solution to pH6.5 followed by the addition of formaldehyde and then titrating to pH9.0. 5. The three methods have been applied to carbamoylated and benzenesulphonylated gelatin derivatives. The values for the degrees of substitution obtained by the ninhydrin (alpha-amino plus in-amino) and the formol (pH9.0, in-amino) methods were shown to be almost identical. The values from the formol (pH6.5-9.0, alpha-amino plus in-amino plus imidazole) titration were consistently lower than the values obtained by the other methods and corresponded to no substitution having taken place at the imidazole groups by the preparative methods employed. 6. The application of the methods to other protein systems is discussed.