Electron paramagnetic resonance study of intersubunit interactions in nitrosyl-deoxy asymmetrical hybrid haemoglobin

Abstract

The asymmetrical nitrosyl-deoxy hybrid haemoglobin, ( α NO β NO), ( α deoxy β deoxy), was prepared by removing oxygen with sodium dithionite from a mixture of oxyhaemoglobin and nitrosylhaemoglobin (Cassoly, 1978). This asymmetrical hybrid exhibited a distinctive triplet hyperfine structure in the electron paramagnetic resonance spectrum. This triplet has been shown to arise predominantly from the nitrosyl haem of an α subunit which has a deoxy-like structure (Nagai et al., 1978). By removing one or two carboxyl-terminal residues by carboxypeptidase digestion before mixing, one can obtain asymmetrical nitrosyl-deoxy hybrid haemoglobins in which only one of the four subunits is specifically modified. Eight such modified derivatives were examined by e.p.r. † † Abbreviations used: e.p.r., electron paramagnetic resonance; Hb, HbO 2, HbNO, and deoxyHb are haemoglobin, oxyhaemoglobin, nitrosylhaemoglobin and deoxyhaemoglobin, respectively. [HbNO and deoxyHb] indicates a solution of HbNO and HbO 2 that was mixed and allowed to react for a few minutes and then deoxygenated by addition of sodium dithionite. ( α M β M) ( α N β N) represents an asymmetrical hybrid haemoglobin tetramer. M and N indicate the state of ligation. A dimer in parentheses means an α 1 β 1 dimer as defined by Perutz (1965). desArg, the removal of Arg(141α); desArg-Tyr, the removal of Arg(14α)Tyr(140α):desHis, the removal of His(146β); desHis-Tyr, the removal of His(146β)Tyr(145β). These 4 prefixes are used to define the modification of subunits in Hb; for example, desArg Hb means Hb with 2 Arg(141α) removed from 2 α subunits, and (desArg-Tyr α NO β NO) ( α deoxy β deoxy) means nitrosyl-deoxy asymmetrical hybrid with Arg(141α) and Tyr(140α) removed from the nitrosyl α subunit. IHP, inositol hexaphosphate. . They were (desArg α NO β NO) ( α deoxy β deoxy), (desArg-Tyr α NO β NO) ( α deoxy β deoxy), ( α NOdesHis β NO) ( α deoxy β deoxy), ( α NOdesHis-Tyr β NO) ( α deoxy β deoxy), ( α NO β NO) (desArg α deoxy β deoxy), ( α NO β NO) (desArg-Tyr α deoxy β deoxy), ( α NO β NO) ( α deoxydesHis β deoxy) and ( α NO β NO) ( α deoxydesHis-Tyr β deoxy), where desArg, desArg-Tyr, desHis and desHis-Tyr indicate that the amino acids were removed from the carboxyl terminus of the subunit. The e.p.r. spectra for these eight derivatives have a more or less reduced relative intensity of the triplet, indicating that the non-covalent bonds involving carboxyl-terminal residues which stabilize the structure of deoxyhaemoglobin (Perutz, 1970) must all be intact in the unmodified asymmetrical nitrosyl-deoxy hybrid haemoglobin, ( α NO β NO) ( α deoxy β deoxy). By comparing the relative intensity of the triplet we were able to examine the effect of modification of one specific carboxyl terminus on the nitrosyl haem in the α 1 subunit. The effect was not symmetric, but increased in the order α 1 < β 2 < β 1 < α 1 (suffices 1 and 2 as defined by Perutz (1965)). We attribute this order to the non-equivalence of intersubunit interactions.