HISTOCHEMICAL AZO COUPLING OF PROTEIN HISTIDINE BRUNSWIK'S NITRATION METHOD

Abstract

The xanthoproteic reaction is accomplished with only 20% HNO3 with glacial acetic acid (HAc) as the solvent; in water 40% HNO3 is required. Tyrosine and 3-mononitrotyrosin readily azo-couple in alkaline solution with fresh p-nitrodiazobenzene in vitro; 3,5-dinitrotyrosine does not. In vitro p-nitrodiazobenzene at pH 8.5 does not couple with histidine, tryptophan or tyrosine after overnight nitration in tetranitromethane (TNM)-pyridine-0.1 N HCl 1:20:40. Histochemical nitration of tissue adequate to prevent the p-diazobenzenesulfonic acid, pH 1-0.02% azure A sequence reaction of hair medulla and arterial elastin of man, dog and rodents can be achieved by 40% HNO3 in glacial acetic acid-acetic anhydride (Ac2O) mixtures. Acetic anhydride should be 10% only, to restrict evolution of brown oxides of nitrogen and prevent undue section losses. Exposures of 4 hr at 3°C to 4:5:1 HNO3:HAc:Ac2O are effective and well tolerated. Nitration is also effectively accomplished by 6-hr 25°C exposures to mixtures of 1% tetranitromethane-pyridine and two volumes of water (pH 8.2) or 0.1 N HCl (pH 6.6). Even 10% TNM in dry pyridine and 2.5% in 16.3% pyridine alcohol (6 hr), 3°C exposures gave only partial tyrosine blockades. The pyridine-water TNM mixtures also prevented the Morel-Sisley tyrosine reaction and, with greater exposures, the postcoupled benzylidene indole reaction of tryptophan as well. Sites reactive to diazobenzenesulfonic acid-azure A after the best nitrations are probably assignable largely to histidine, though the presence of some unblocked tryptophan, purines and other reactive substances must be considered.