Carbon Monoxide Religation Kinetics to Hemoglobin S Polymers following Ligand Photolysis

Abstract

The re-equilibration rate of carbon monoxide binding to hemoglobin S polymers is determined by time-resolved measurements of linear dichroism spectra. Linear dichroism is used to detect religation to hemoglobin in the polymer in the presence of rebinding to free hemoglobin S tetramers. Measurement of the linear dichroism resulting from photolysis of the small percentage of ligand bound to the polymer is accomplished through the use of an ultrasensitive, ellipsometric linear dichroism technique developed for this purpose. The major finding is that the return of the polymer phase to its equilibrium ligation state is much slower than that of the solution phase hemoglobin tetramers. Assuming all hemes in the polymer are equally likely to participate in rebinding, the re-equilibration rate for carbon monoxide religation to hemoglobin S polymers is found to be 0.14 ؎ 0.07 (s ؊1 mM ؊1 ), about 1000 times slower than the rebinding rate of carbon monoxide to T-state monomer hemoglobin. Several interpretations of this result are discussed. An understanding of the ligand binding kinetics to hemoglobin S polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.

Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility

“…For the deoxyHbS and partially nitrosylated HbS samples, one can flip the LD signal (as shown) by rotating the sample about the axis defined by the incident light. The ability to flip the signal is strong evidence that it results from LD of partially aligned polymers (27). Fig.…”

A Study of the Mechanisms of Slow Religation to Sickle Cell Hemoglobin Polymers Following Laser Photolysis

“…Using known CO on rates for R and T state hemoglobin one predicts that CO recombination is complete by 10 ms after photolysis (Bertini et al, 1994;Shapiro et al, 1995). It then seems impossible that any CO would be left to rebind the polymer a second after photolysis.…”

“…The linear dichroism from the molecules in the polymer phase disappears with the kinetics of religation because the polymers do not rotate. We reported previously that religation of the polymer phase occurs about 1000 times slower than that of the monomer phase (Shapiro et al, 1995). We proposed several mechanisms for this slow religation: (1) religation occurs through a bimolecular process involving all polymer hemes (henceforth referred to as the bimolecular mechanism), (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate in polymer religation (ends mechanism), and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase (exchange mechanism).…”

“…Using time-resolved linear dichroism (TRLD) measurements, we have reported the religation kinetics of CO to HbS polymers following laser photolysis (Shapiro et al, 1995). In these experiments, the liner dichroism is measured at precise times after CO has been photolyzed by a polarized actinic beam.…”

“…Another potential mechanism involves dissociation of CO from solution phase hemoglobin molecules followed by binding of these CO molecules to polymer phase hemoglobin. This mechanism involving CO dissociation was excluded as an explanation for polymer religation because the CO dissociation rate is too slow to account for the observed religation lifetimes (Shapiro et al, 1995).…”