Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

Abstract

Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include pre-equilibria of tertiary as well as quaternary conformations; the structure-based model of Szabo and Karplus; and the modification of the latter model by Lee and Karplus. We show that only the tertiary two-state model can provide a near quantitative explanation of the single-crystal and gel experimental results.

Treating sickle cell disease by targeting HbS polymerization

“…Moreover, the sickle fiber binds oxygen with an affinity that is very similar to the T quaternary structure in the crystal. 69,70 These results are consistent with structural analysis, which shows that the arrangement of the subunits in the R structure produces many steric clashes 71 that preclude incorporation into the T-containing fiber.…”

Half a Century of Hemoglobin’s Allostery

“…Over and above presenting their data, Henry et al (9) discuss the TTS model by comparison with previous articles, and particularly the statistical mechanical description of Perutz's stereochemical mechanism published by Szabo and Karplus (10).…”

“…Perutz et al (6) had discovered that deoxy human Hb, crystallized from polyethylene glycol at low pH, maintains the salt bridges intact even upon ligand saturation. Accurate oxygen binding curves of these crystals obtained by microspectrophotometry (Henry et al (9) and references therein) showed that 1) the saturation curve is noncooperative (Hill's coefficient n ¼ 1); and 2) the oxygen affinity is very low and independent of pH, BPG, and other effectors. These findings are consistent with predictions of the MWC model whereby stabilization of the T state is associated to low affinity, and inconsistent with the Koshland, Nemethy, and Filmer-induced fit mechanism.…”

“…Although the physical state of the protein in the cavity is by-and-large a conundrum, both entrapped allosteric states are stable for hours to days and display noncooperative oxygen binding with either low affinity (the T state) or high affinity (the R state). The silicagel locking protocol has been exploited successfully by the Bethesda-Parma group (Henry et al (9) and references therein). They confirmed that R-and T-state Hb locked-in silica gels bind oxygen noncooperatively; however, at variance with data with the crystals (see above,) oxygen affinity of entrapped T-state Hb is sensitive to pH and BPG as the protein in solution (12).…”

“…The article by Henry et al (9), offered in this issue of the Biophysical Journal, discusses the experimental basis for proposing the ''simplest possible extension of the MWC model'', a variant termed by them the tertiary two-state (TTS) model, which includes tertiary as well as quaternary pre-equilibria. Over and above presenting their data, Henry et al (9) discuss the TTS model by comparison with previous articles, and particularly the statistical mechanical description of Perutz's stereochemical mechanism published by Szabo and Karplus (10).…”