Nuclear relaxation and gelation study of the interaction of organophosphates with human normal and sickle hemoglobins. In vitro gelation of sickle oxyhemoglobin in the presence of inositol hexaphosphate.

31P relaxation studies reveal a 3-fold enhancement of the longitudinal relaxation rate of both phosphoryl groups of hemoglobin-bound 2,3-bisphosphoglycerate upon conversion of methemoglobin to fluoromethemoglobin presumably due to an order of magnitude increase in the electron spin relaxation time. The enhancement of the longitudinal components of 31P relaxation (T(-1)1pr) upon binding to hemoglobin is not exchange-limited, since it is more than an order of magnitude smaller than the effect observed on the transverse components (T(-1)2pr). From the observed paramagnetic component, T(-1)1M, of the bound state relaxation rate of 2,3-bisphosphoglycerate, using the correlation time tau s obtained from the frequency dependence of water proton relaxation, we obtained an NMR root mean sixth distance from the four heme iron atoms to each of the 31P nuclei of 24 +/- 1 A. This is in excellent agreement with the x-ray crystallographic determination of this distance of (25 +/- 1) A in the 2,3-bisphosphoglycerate-deoxyhemoglobin complex, indicating that the spatial disposition of the allosteric site in the deoxy and oxy conformations of hemoglobin relative to the various heme irons may be the same, and that the same protein groups may be involved in binding 2,3-bisphosphoglycerate to the two forms of hemoglobin. Water proton relaxation studies reveal the existence of different conformational states of methemoglobins with 2,3-bisphosphoglycerate and inositol hexaphosphate. Inositol hexaphosphate alters the conformation to a strained (T) state with deoxy-like quaternary and tertiary globin structure as indicated by the finding that equimolar amounts of inositol heasphosphate induce gelation in a 4 mM sickle methemoglobin solution at temperatures greater than or equal 24 degrees. More interestingly, oxyhemoglobin S shows an identical thermodynamically reversible gelation behavior, with the same transition temperature (24 degrees), arguing against a mutual coupling of the protein conformation and the heme spin state in functional ferrohemoglobins. High ionic strengths (approximately 1 M) and pH values above neutrality block inositol hexaphosphate induced gelation of sickle met- and oxyhemoglobins. Unlike inositol hexaphosphate, the presence of saturating amounts of 2,3-bisphosphoglycerate does not promote gelation of a 4 mM met- or oxyhemoglobin S solution.