States of Amino Acid Residues in Proteins:XVI. Naphthoquinones with two Sulfonic Groups as a Reagent for Discrimination of Amino Groups in Proteins

The reagents so far used for partial modification of amino groups in proteins give rise to modified proteins which are unstable in aqueous solutions because of the lowering of solubility by the modification. In these circumstances, β-naphthoquinone-4, 5-, -4, 6- and -4, 7-disulfonic acids were examined as reagents for modification of amino groups and applied to several proteins, and the following results were obtained. The 4, 6- and 4, 7-disulfonic derivatives possess a moderate reactivity with amino groups near neutral pH (optimum pH between 8.4 and 9.1) and at room temperature, which is suitable for discrimination of different states of amino groups in proteins, and yet the proteins modified with these reagents are stable in solution. On the other hand, the 4, 5- disulfonic derivative is not reactive with amino groups under the same conditions. The degree of reaction can be determined simply by difference spectrophotometry at 480mμ between the sample and blank solutions. Approximately 2 of the total 3 amino groups in the insulin molecule and 9 of the 11 amino groups in the ribonuclease [EC 2. 7. 7. 16] molecule are reactive with the 4, 6- or 4, 7-disulfonic derivatives. About 6 of the total 7 amino groups in the lysozyme [EC 3. 2. 1. 17] molecule are modified with these reagents and the lytic activity is decreased by the modification to less than 3% of the original activity. In the case of trypsin [EC 3. 4. 4. 4], 25-40% of the activity is retained even after modification of almost all of the amino groups. Treatments of two samples of α-chymotrypsin [EC 3. 4. 4. 5] purchased from different companies with the reagents gave different results ; 14 and 17 amino groups per molecule are modified with the reagents and the activity drops to 60% and less than 6% on the modifications, respectively. At least one of the amino groups was inferred from these data to participate in the enzyme activity.