The Reaction of Hemoglobin with Nitrite

Although nitrite has been extensively used as an agent for the formation of methemoglobin, the quantitative relations of the reaction appear to be uncertain; three widely different values have been reported for the amount of methemoglobin formed to nitrite utilized. The first statement of a quantitative relationship was made by Barcroft and Miiller (1) in 1911. In a preliminary report containing no experimental data or equations, and for which none was supplied in subsequent publications, they indicated the molar ratio of nitrite utilized to methemoglobin formed as 2; that is, 2 molecules of nitrite react with 1 molecule of hemoglobin to produce 1 molecule of methemoglobin. This relation was accepted by Stadie (2) in 1921 in his investigations on methemoglobin formation. From experimental determinations, Van Slyke and Vollmund (3) in 1925, Meier (4) in 1925, and von Issekutz (5) in 1939 reported that 1 molecule of nitrite was used in forming 1 molecule of methemoglobin; i.e., a molar ratio of 1. Austin and Drabkin (6) in 1935 reported a ratio of 0.5 to 0.7 and one of approximately 0.5 can be calculated from data published in 1942 by Darling and Roughton (7) dealing with the effect of methemoglobin on the oxygen dissociation curve. The reasons for some of these discrepancies will be dealt with in detail in subsequent discussion. An obvious source of error in several of the investigations was in the assumption that all nitrite added to blood or solutions of hemoglobin was quickly and completely utilized in methemoglobin formation. Analyses were not made for residual nitrite. In the investigation reported here, this source of error was avoided. The amount of nitrite reacting with hemoglobin was determined and the influence of temperature, concentration of nitrite, and of hydrogen ion concentration on the reaction was studied.