Physical Basis of the Effect of Hemoglobin on the³¹P NMR Chemical Shifts of Various Phosphoryl Compounds

Abstract

The marked difference between the intra- and extracellular31P NMR chemical shifts of various phosphoryl compounds when added to a red cell suspension may be largely understood in terms of the effects of hemoglobin on the31P NMR chemical shifts. The presence of [oxy- or (carbonmonoxy)-] hemoglobin inside the red cell causes the bulk magnetic susceptibility of the cell cytoplasm to be significantly less than that of the external solution. This difference is sufficient to account for the difference in the intra- and extracellular chemical shifts of the two phosphate esters trimethyl phosphate and triethyl phosphate. However, in the case of the compounds dimethyl methylphosphonate, diethyl methylphosphonate, and trimethyl-phosphine oxide as well as the hypophosphite, phenylphosphinate, and diphenylphosphinate ions, hemoglobin exerts an additional, much larger, effect, causing the31P NMR resonances to shift to lower frequency in a manner that cannot be accounted for in terms of magnetic susceptibility. Lysozyme is a protein structurally unrelated to hemoglobin and was shown to cause similar shifts to lower frequency of the resonances of these six compounds; this suggests that the mechanism may involve a property of proteins in general and not a specific property of hemoglobin. The effect of different solvents on the chemical shifts of the eight phosphoryl compounds provided an insight into the possible physical basis of the effect. The31P NMR chemical shifts of the two phosphate esters were entirely insensitive to the nature of the solvent whereas those of the other six compounds showed a very strong solvent dependence which is understood, primarily, in terms of the number and strength of hydrogen bonds formed between the solvent molecules and the phosphoryl oxygen atoms. It is therefore proposed that, in addition to magnetic susceptibility effects, hemoglobin exerts its influence on phosphoryl chemical shifts by disrupting the hydrogen bonding of the phosphoryl group to solvent water.