Comparison of histidine proton magnetic resonances of human carbonmonoxyhaemoglobin in different buffers

Abstract

We have recorded the C-2 proton resonances of the histidines of carbonmonoxyhaemoglobin A and of four abnormal human HbCOs^† in different buffers and at different concentrations of haemoglobin. Resonance H assigned by Perutz et al. (1985) to His HC3(146)β, is present at both pH 7.30 and pH 6.90, but somewhat broadened when recorded in 5 to 10% HbCO A in 0.1 m-bis-Tris. The broadening disappears on tenfold dilution of the Hb with bis-Tris and the resonance then stands out sharply. Resonance H is absent at both Hb concentrations in HbCO Cowtown (His HC3(146)β → Leu). HbCO Fort de France (His CD3(45)α → Arg) in 0.1 m-bis-Tris of pH 6.90 has a spectrum similar to that of HbCO A. In the same buffer a resonance marked L by Russu et al. (1982) is absent from the spectrum of Hb Abbruzzo (His H21(143)β → Arg), whereas resonance H is present. Hb Barcelona contains an additional histidine in position FG 1(94)β; in 0.1 m-bis-Tris buffer of pH 6.90 its resonance is not resolved and resonance H is either shifted or broadened. The resonances of both histidines are resolved in phosphate buffer. At pH 6.90, spectra in 0–1 m-bis-Tris buffer are similar to those previously recorded in 0.2 m-HEPES. Addition of 0.1 m-KCl produces marked changes. Replacement of bis-Tris by 0.2 m-KCl + 0.2 m-phosphate gives rise to a different and much better resolved spectrum.