Cross correlation of titrating histidines in oxy‐ and deoxyhaemoglobin; an NMR study

Abstract

Haemoglobin is the best known example of a protein exhibiting co-operative binding properties and continues to excite active interest [1][2][3]. One feature of the co-operative behaviour is the release of protons on binding oxygen, a phenomenon known as the Bohr effect. Perutz in his classic X-ray work [4] identified three groups which might be involved in the Bohr effect and Kilmartin et al. [5] in an n.m.r. study of CO and deoxy forms of haemoglobin confirmed that one of these groups was His/3146. Val al has also been identified, by chemical modification studies [6], as another group involved but there is still uncertainty about the role of other groups, although these are expected to be histidines. N.m.r. seems to be the technique most likely to provide the detailed information about ionization states necessary to resolve this problem, but at present it is limited by difficulties in resolving and assigning the titrating histidine resonances.The key factor in an n.m.r, study of the involvement of histidines in the Bohr effect lies in the correlation of peaks in oxy-and deoxyhaemoglobin. In this communication we illustrate a powerful method for correlating peaks whose movements in an oxydeoxy titration are discontinuous and therefore impossible to follow by stepwise titration procedures. The method, which has been used previously to correlate resonances of reduced and oxidised cytochrome c [7] requires that the rate of exchange between liganded and unliganded forms be faster than the nuclear spin lattice relaxation rate (l/T1). This requirement is shown to be met for at least one histidine resonance in haemoglobin when oxygen is the ligand.

Inhibition of Papain by N‐Acyl‐aminoacetaldehydes and N‐Acyl‐aminopropanones

“…Although cross-saturation in NMR spectroscopy has been successfully applied to correlate resonances in the oxidised and reduced states of cytochrome c [22], in the oxy and deoxy forms of haemoglobin [23], and in the aromatic protons of a slowly rotating tyrosine residue in ferrocytochrome c [24], as far as we are aware this is the first application of the technique for the investigation of an enzyme-inhibitor interaction. The technique appears to have considerable potential for the detection of intermediates in enzymecatalysed reactions.…”

Structure-specific model of hemoglobin cooperativity.

“…1) there are four salt bridges per aB dimer (2-4, 23), of which two titrate in the physiologic pH range. Supporting evidence for the identification of the Bohr groups has come from NMR (24,25), from hydrogen exchange experiments (26), from chemical reactivity studies (27,28), and from studies of mutant and modified hemoglobins (29)(30)(31)(32). Measurements indicate that the a1-a2 and intra-,B-chain Bohr groups contribute 60-80% of the total alkaline Bohr effect at normal (e.g., 0.1 M Cl-) salt concentrations (20,(29)(30)(31)(32)(33)(34).…”

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

“…To circumvent this problem, we have identified the position of the resonance of the bound coenzyme by saturation transfer experiments (Forsén & Hoffman, 1963;Red field & Gupta, 1972;Campbell et al, 1976Campbell et al, , 1977Brown & Campbell, 1976;Bendall et al, 1977;Chen et al, 1979;Cayley et al, 1979).…”