CO Rebinding Kinetics to Myoglobin- and R-State-Hemoglobin-Doped Silica Gels in the Presence of Glycerol

Abstract

This study characterizes the CO rebinding kinetics after photodissociation of horse heart myoglobin (Mb) and human R-state hemoglobin (Hb) encapsulated in wet silica gels, in the presence of various concentrations of glycerol. The geminate yield for HbCO is scarcely affected by the gel matrix, indicating that the protein can fluctuate as in a homogeneous solution. On the contrary, the geminate yield for gel-embedded MbCO is much higher than that in solution, suggesting that the gel matrix inhibits the movements of the protein. The geminate yield for both proteins increases substantially with the addition of glycerol to the bathing solution. The observed kinetics could be rationalized using a simple three-state model. Rate constants have been modeled using a modified Kramers equation, which indicated that the gel exerts an internal friction on the elementary rate constants. The rate constant for geminate rebinding, k CA, is essentially viscosity independent below ∼20 cP for both proteins. The internal friction for the ligand escape rate, k CS, is much smaller and is found to be negligible for HbCO and ∼5 cP for MbCO. The activation barrier for k CS increases with glycerol concentration in response to increased viscosity and reduced ligand solubility. The rate k SC showed a complex behavior that reflects the opposing effects of viscosity and activity arising from molecular confinement and crowding. Accordingly, the corresponding activation barriers show a biphasic behavior, with a minimum at ≈40% glycerol for HbCO and at ≈75% glycerol for MbCO. The results highlight the potential of silica gel encapsulation for in vitro studies aimed to reproduce the crowded and confined environment experienced by proteins in vivo. The diverse response to encapsulation of Mb and Hb could actually reflect physiologically relevant functional properties escaping detection in the diluted solutions normally used for biophysical investigations.

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

“…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. …”

“…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).…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…When studying protein dynamics and conformational equilibria in vitro, it should be considered that caging effects in the pores of the silica gel matrix might help mimic crowding and confinement experienced in vivo by proteins [66,102,103] much better than the diluted solution normally used for biochemical and biophysical investigations [9,46,65,98,[104][105][106][107]. Silica gel encapsulation was exploited to investigate caging effects on the unfolding mechanism of a highly fluorescent variant of Aequorea victoria Green Fluorescent Protein (GFP), GFPmut2 (S65A, V68L, S72A GFP) [108].…”

“…In particular, it has been shown that after photolysis of HbCO encapsulated in the T state, both the E and F helices assume intermediate positions with respect to those that characterize the Hb equilibrium states. Moreover, ligand rebinding kinetics of CO to encapsulated Hb were measured in flash photolysis experiments both at room [58,87,89,90,127,[129][130][131][132] and cryogenic temperatures [88]. Inhibition of the quaternary transition allowed to study CO rebinding kinetics to pure T and R states.…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…The differential equations corresponding to ligand migration in Cygb ox can be solved analytically as previously reported by Sottini et al (2004) for hemoglobin and the microscopic rate constants can be calculated according to equations 3.8 to 3.13. The overall geminate rebinding rate (k gem )…”