Sol−Gel Trapping of Functional Intermediates of Hemoglobin:  Geminate and Bimolecular Recombination Studies

Abstract

The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint 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 sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from 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 pulsed laser sources and that one of the tested encapsulation protocols is far 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.

Ligand recombination and a hierarchy of solvent slaved dynamics: The origin of kinetic phases in hemeproteins

“…The hygroscopic nature of glycerol makes it difficult to prepare glycerol bathed samples that are all identical with respect to residual water content. Since the glycerol/water ratio has been shown to have clear effect on the relative amplitudes and onset of the kinetic phases (Khan et al, 2000;Khan et al, 2001;Sottini et al, 2004;Sottini et al, 2005a;Sottini et al, 2005b;Sottini et al, 2005c), it is possible that anticipated small variations in water content in different glycerol bathed sol-gel samples may influence the time points for the onset of the C and D state dynamics. There are also clear indications that the templated environment surrounding the encapsulated protein differs from that of bulk solvent in the pores of sol-gel that link the interior to the exterior of the sol-gel (Massari et al, 2006).…”

“…These would include for Mb, the dynamics contributing to the relaxation of the initial photoproduct tertiary structure back to the equilibrium deoxy conformation and for hemoglobins the additional larger scale motions required for changes in quaternary structure. Both categories of dynamics can be greatly slowed within sol-gel matrices relative to the operative time scales of the C and D tiers of dynamics (Bettati and Mozarrelli, 1997;Das et al, 1999;Khan et al, 2000;Abbruzzetti et al, 2001;Dantsker et al, 2002;Samuni et al, 2002;Shibayama and Saigo, 2003;Samuni et al, 2004;Viappiani et al, 2004). Consequently, the above A, B, C and D scheme can be explored within the conformational boundaries of different trapped tertiary and quaternary structures that represent both equilibrium and nonequilibrium conformational distributions.…”

“…The present treatment represents a distillation and out growth of our recent work utilizing sol-gel encapsulation and glassy matrices to modulate protein dynamics in a way that provides a direct bridge between cryogenic and ambient domains Dantsker et al, 2005a;Dantsker et al, 2005b). We (Khan et al, 2000;Dantsker et al, 2002;Samuni et al, 2002;Dantsker et al, 2005a;Dantsker et al, 2005b) and others (Sottini et al, 2004;Sottini et al, 2005a;Sottini et al, 2005b;Sottini et al, 2005c) have shown that this approach of using high viscosity solvents and matrices exposes the dynamical events that give rise to the solution phase functional properties observed in solution at ambient temperatures. …”

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”

Nanoparticles as a Novel Delivery Vehicle for Therapeutics Targeting Erectile Dysfunction

“…It is well established that these types of nanoparticles provide excellent matrices for encapsulating a wide variety of organic and inorganic compounds, including many biologically relevant materials, such as proteins [4][5][6], NO [3], and pharmaceuticals [7]. The resulting material can be converted into powders comprised of nanoparticles with average diameters in the range of tens of nanometers (for comparison, viruses are 10-300 nm or eukaryotic cells 2-100 μm in diameter).…”