States of Amino Acid Residues in Proteins XII. Amino Groups with Different Reactivities toward Naphthoquinone Sulfonic Acid

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Abstract
β-Naphthoquinone-4-sulfonic acid (NQS) was examined and applied to several proteins as a reagent for discriminating various states of amino groups in the tertiary structures of proteins, and the following facts were revealed. NQS possesses a moderate reactivity suitable for the discrimination at room temperature and between pH 7.5 and 9.3 where most proteins are native, and the degree of reaction can be determined simply by absorption photometry at 480 mμ Four of the total 7 amino groups in the lysozyme [EG 3.2.1.17] molecule, 7 of the 11 amino groups in ribonucleasc [EC 2.7.7.16], 14 of the 17 groups in α-chymotrypsin [EC 3.4.4.5], 12 of the 15 groups in chymotrypsinogen and 12 of the 17 groups in diisopropylphosphoryl-chymotrypsin are more reactive with NQS than the remaining 3, 4, 3, 3 and 5 groups, respectively. Two terminal amino groups of the A and B chains of insulin are reactive, and the remaining ε-amino group of B 29 Lys is not reactive with this reagent. The non-reactive-amino group is transformed into the reactive type by alkali denaturation but is not transformed by tryptic digestion. It was deduced from these results that the amino group is hydrogen-bonded with a residue in the heptapeptide released by the digestion. The counterpart in the hydrogen-bonding was inferred to be the hydroxyl group of B26 Tyr or B27 Thr.

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