Sol-gel trapping of functional intermediates of hemoglobin: Geminate and bimolecular recombination studies

The encapsulation of proteins in porous sol-gels is a promising technique f or generating, trapping, and probing functionally significant nonequilibriu m protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change u pon adding or removing substrates. In the present study, geminate recombina tion and solvent phase bimolecular recombination of CO to human adult hemog lobin (HbA) are used as sensitive probes of the degree of conformational co nstraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO , the equilibrium form of deoxyHbA is encapsulated and only after the sampl e has aged is CO introduced to the HbA through the porous sol-gel matrix. T hree different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained fr om these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pu lsed laser sources and that one of the tested encapsulation protocols is fa r superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.