Histochemical azo coupling of protein histidine. Brunswik's nitration method.

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% tetra...