Reexamination of the evidence for paramagnetism in oxy- and carbonmonoxyhemoglobins.

Abstract

Experiments have been carried out in an attempt to confirm previous reports of paramagnetism in the oxy-and carbonmonoxy derivatives of human and carp hemoglobin. When care is taken to ensure complete saturation of the hemoglobins with ligand and the diamagnetic contributions of all of the buffers are carefully evaluated, these hemoglobin derivatives are found to have the same gram susceptibilities as exhibited by a set of metal free proteins, suggesting that they are fully diamagnetic.In previous articles (1-5), we reported evidence of the existence of a paramagnetic contribution to the magnetic susceptibilities of the carbonmonoxy and oxy derivatives of human and carp hemoglobins. In response to the controversy surrounding our observations and the findings of others (6, 7), we have repeated the experiments with additional controls. We wish to report that there is no longer any reason to suspect that these derivatives are paramagnetic at room temperature. Using a few metal-free proteins we have been able to establish a diamagnetic reference for protein solutions. We find that the magnetic properties of oxy-and carbonmonoxyhemoglobins correspond to such a diamagnetic reference. This brings our observations into agreement with those of refs. 6 and 7.The methods of sample preparation, the instrumentation, the overall precision, and the data reduction were the same as in our most recent papers (see ref. 5). However, we took greater care to be certain that the hemoglobins were fully saturated with the ligand in the highly concentrated solutions used in those experiments. We did this after some observations of our own and discussions with Joe Savicki and George Lang aroused the suspicion that partial deoxygenation could have been the source of our previous findings. For every protein sample the diamagnetic contribution of the buffer was determined independently. These determinations led us to revise the values of the buffer corrections at high salt concentrations. Our results are shown in Fig. 1 Fig. 1. We obtain for the common Xg the value -(0.587 + 0.005) x 10-6 ml/g. We have also measured the susceptibilities of solutions of human HbO2 and HbCO and carp HbCO at high salt concentrations (-350 mM NaCl). The Xm. values for all of these derivatives fall, within error limits, on the same straight line when plotted as a function of molar heme concentration with the intercept at zero concentration equal to the solvent Xm., Using the same value for the partial volume of Hb as that used for the low-ionic- strength results, we calculate XHb to be -(0.593 + 0.006) x 10-6 ml/g. It is evident that no significant difference exists between XHb at low and high salt concentrations. This value is consistent with our earlier results obtained with human HbCO at low salt concentrations but not with the results we reported for this derivative at high ionic strength in ref. 3. Finally, we have also redone our studies in the frozen state. Fully liganded human HbCO and HbO2 samples were frozen, by immersion in liquid nitrogen, and...

Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility,T1,T2,T, and non‐Lorentzian signal behavior

“…Note that it has been shown that a variety of nonparamagnetic metal-containing and metal-free proteins exhibit the same value of g ϭ -0.587 ϫ 10 -6 mL/g (30). With the use of Eq.…”

“…[1], Y is the fraction of hemoglobin that is present in the form of oxyHb, n Hb is the total intracellular Hb concentration, 5.5 ϫ 10 -6 mol/mL (29), and g,protein is the gram susceptibility, diamagnetic contribution of the Hb protein which is equal to -0.587 ϫ 10 -6 mL/g (30). Other parameters included in the above equation are the volume susceptibility of water, H2O ϭ -0.719 ϫ 10 -6 (31), the molecular weight of deoxyHb, M Hb ϭ 64,450 g/mol (32) and the molar volume of Hb in solution, MHb ϭ 48,277 mL/mol (28).…”

Quantitative susceptibility mapping‐based cerebral metabolic rate of oxygen mapping with minimum local variance

“…Here, 0:587 Â 10 À6 mL=g is the mass susceptibility of globin relative to vacuum in CGS units (53,54); 4p is the unit conversion from CGS to SI units; and À9035 ppb is the water volume susceptibility relative to vacuum (55). X p ¼ À37:7 ppb is the oxyhemoglobin and blood plasma volume susceptibility (12).…”

Discovery of the magnetic behavior of hemoglobin: A beginning of bioinorganic chemistry

“…Pauling insisted that this result could not be correct (39), as did others (40), but the Italian group reported additional experiments at room temperature in support of their model (41). In 1985, however, Cerdonio threw in the towel (42). One of us (H.B.G.)…”