A proton magnetic relaxation study of human ferrihaemoglobin in aqueous salt solutions

Abstract

SynopsisThe proton magnetic relaxation time, 2'1, has been measured at 29 MHz in 0.1M KH2P04 and 0.1M NaCl (both pH 6 ) aqueous solutions of human ferrihaemoglobin, the protein concentrations ranging from 0.5 to 5 mM per haem. The linear dependence on protein concentration of the enhancement in relaxation rates, A(l/Tl), due to the presence of the paramagnetic iron in haemoglobin was confirmed at 34°C and a t -10°C. In the middle temperature range there is a thermally activated process, whose energy of activation depends on protein concentration. This dependence is different for the two salt solutions; E , increases with CHb for O.1M KH2POr and decreases for 0.1M NaCl.The model of water-proton exchange between the bulk solvent and the sixth coordination site of the haem iron was used to calculate the distance from the "liganded" water protons to the haem iron. This yields distances much larger than those determined by X-ray crystal structure analysis. A model is proposed which reconciliates both types of data. The low-temperature relaxation rates cannot be used in deriving quantitative stereochemical data for the haem pocket because of its special shape.Irrespective of the molecular model adopted, the experimental results show clearly that, both a t low (-1OOC) and higher (>34"C) temperatures, the interaction of paramagnetic haem iron with water protons is practically the same for the two aqueous solutions. The dynamic state of the haemoglobin molecule, as indicated by the middle-temperature range, is completely different in 0.1M KH2POl and 0.144 NaC1, pH 6.

Interactions of solvent with the heme region of methemoglobin and fluoro-methemoglobin

“…The major result is that outer sphere relaxation accounts for a substantial part of the observed solvent proton relaxation rate. At low fields and 60C, it is about two-thirds of the total at all values of pH considered; at 350C it is less, consistent with a contribution from thermally activated exchange of a water molecule from the inner sphere (Pifat et al, 1973). Moreover, as mentioned, these are lower limits.…”

“…These authors, as remarked above, ascribed all the relaxation to outer sphere effects. Pifat et al (1973), in an extensive investigation of the temperature dependence of proton relaxation in solutions of methemoglobin, also concluded ". .…”

Quantitative theory for the longitudinal relaxation time of blood water

“…Although initial studies about the paramagnetic relaxation in methemoglobin attributed the relaxation enhancement to the “inner sphere” mechanism, i.e., only water ligated with heme relaxes fast, and then exchange with bulk water. However, an extensive investigation of proton relaxation dependence on temperature had shown that the exchange rate between ligated water and bulk water was not fast enough to induce the fast relaxation, and Koenig et al measured the relaxation dispersion curve at multiple magnetic fields and proved the “outer sphere” mechanism, i.e., the fluctuation of the dipolar coupling between water and paramagnetic protein due to water diffusion, dominated the paramagnetic relaxation. Therefore, Freed's diffusion model which described water molecules diffusing around the paramagnetic complex was used to fit the field dependence of the paramagnetic relaxivities of methemoglobin and deoxyhemoglobin in Figure .…”

Frequency dependence of the proton magnetic relaxation in aqueous solutions of haemoproteins

“…Bovine haemoglobin was prepared as described previously. 6 The final dialyses were against 0.1 M phosphate buffer, pH 6, with the addition of Horse metmyoglobin from Koch and Light was used without further purification. The lyophilized powder was dissolved in the same buffer and centrifuged a t 14000 g for 20 min.…”