The combining power of normal human hemoglobin for nitrosobenzene.

The nitrosobenzene complex of hemoglobin is a violet pigment which is associated with nitrobenzene poisoning. Filehne (1) reported in 1878 that “nitrobenzol” (nitrobenzene) exerts its toxic effect by making blood unable to transport oxygen. This took place because nitrobenzene apparently underwent a reduction to form a hemoglobin complex with a characteristic spectroscopic absorption which did not correspond to any of the ordinary hemoglobin products. The violet pigment was also observed by Loeb et al. (2) in 1921 while investigat.ing nitrobenzene poisoning. These workers reported that the violet hemoglobin compound had a markedly diminished oxygen capacity. Jung (3) and Keilin and Hartree (4) reported on the reaction of hemoglobin with nitrosobenzene: they showed that a well characterized violet compound was formed with hemoglobin; the same compound was formed from oxyhemoglobin and from carbonmonoxyhemoglobin with the evolution of stoichiometric amounts of 0% and CO, respectively. The latter reaction was reversed by high CO pressure; the compound was quite st.able, and had a broad absorption band like that of reduced hemoglobin but with two weak maxima at approximately 567 and 543 rnp. No quantitative measurements of apparent equilibrium constants were report.ed. During the course of investigations on heavy metal binding sites (predominantly mercapto groups) of hemoglobin and on the nature of the mercapto-mercapt.0 interactions (5) it became necessary to study the influence the presence of a large ligand bound to heme groups might have on the titratability of the mercapto groups as well as on the SH-SH interaction constants. Nitrosobenzene was chosen for the study mainly because of its size, a property common to a number of the well known sulfhydryl reagents, such as p-chloromercuribenzoate and phenylmercuric nitrate.l Furthermore, nitrosobenzene was adequate for the purpose because it is a large molecule with aEinity for the heme groups sufficiently strong to form the complex from oxyhemoglobin. As already stated, its combining constant with hemoglobin was not known. The main object of the study has been to determine the combining constant of human hemoglobin for nitrosobenzene at O”, 25”, and 38” for pH 6.6, 7.0, and 9.0, respectively. Results of this investigation indicate that the hemoglobin binds nitrosobenzene about 6 to 10 times more strongly than oxygen. Most of the data presented concern the binding of