Monitoring the T‐R transition of human hemoglobin encapsulated in silica gels

The entrapment of proteins in wet, nanoporous silica gels allows to stabilize thermodynamically unstable states, thus enabling the characterization of elusive reaction intermediates. In the case of human T and R hemoglobin (Hb) silica gels, the T to R transition of liganded Hb in the gel is dramatically slowed down and allows to study the reactivity towards oxygen or CO of pure T states. Similarly, photolysis of CO complexes of the R state Hb does not induce switching to the T state. Oxygen binding and kinetics of CO rebinding upon laser flash photolysis have supported the Tertiary Two State model, an extension of the classic MWC allosteric model, in which functional properties are associated to changes in the distribution between tertiary t and r states. In the present study, the quaternary transition was monitored by characterizing the CO rebinding kinetics of HbCO gels evolving from T to R, in the absence and presence of allosteric effectors. The analysis with a maximum entropy method indicates that the T state slowly evolves towards the quaternary R state with an associated shift of the distribution of t state towards the r state. The rate of this transition is dependent on the presence of allosteric effectors.