AN IMPROVED SAKAGUCHI REACTION FOR MICROSPECTROPHOTOMETRIC USE

1. The Sakaguchi reaction for arginine has been modified to provide a technique suitable for the quantitative microspectrophotometric estimation of protein arginine. The reagent consists of 2,4-dichloro-α-naphthol, sodium hypochlorite and barium hydroxide. Previous Sakaguchi reagents containing high concentrations of sodium or potassium hydroxide caused extensive damage to the tissue; substitution of barium hydroxide has eliminated this problem. In addition, it appears that barium ions stabilize the resulting Sakaguchi color. 2. Conditions were found for making intensely stained, stable and reproducible preparations which can be dehydrated and mounted to match their index of refraction without loss of colour, and which do not fade significantly for at least two weeks. 3. Intensive acetylation and formalinization, which are thought to block the guanidyl group of arginine, considerably decrease the intensity of the Sakaguchi reaction. 4. The ratio between the amount of Sakaguchi color in bull sperm and in trout sperm, as determined from the results of microspectrophotometric examination, is in rather close agreement with the ratio derived from the amounts of arginine in these cells estimated biochemcially. This supports the concept that the Sakaguchi reaction described here represents a stoichiometric reaction with arginine.