Kinetic and spectroscopic studies of hemoglobin and myoglobin in porous sol -gels

Abstract

Encapsulation of proteins in porous sol-gels is a promising technique both for the development of new classes of biosensors and for biophysical studies of unstable protein conformations. This thesis focuses on the development and the utilization of new encapsulation protocols that trap and lock-in nonequilibrium structures of hemoglobins and myoglobins under conditions where the encapsulated proteins are still functional and subjectable to biophysical characterization. The characterization of encapsulated myoglobin at low pH was studied using resonance Raman spectroscopy. The functional properties of the encapsulated hemoglobin were assessed using photodissociation of carbon monoxide derivatives of encapsulated hemoglobins followed by ligand rebinding kinetics ranging from nanoseconds to hundreds of milliseconds. Conformational properties of the encapsulated proteins were also probed using several optical spectroscopic tools including fluorescence and absorption spectroscopies.;Spectroscopic studies on encapsulated myoglobin, reveal that the sol-gel technique can be used to trap and stabilize the native structure of myoglobin. The gel prevents the rapid unfolding of myoglobin at ambient temperatures. Encapsulation allows for a shift of the pkA of the heme protein such that a lower pH is needed to unfold the native structure. These studies demonstrated that intermediates that are normally difficult to study in solution phase studies were easily isolated in the sol-gel.;A new encapsulation protocol was developed that very successfully locked in and maintained initial conformations despite subsequent changes in the degree of ligand binding. It was observed that when deoxy hemoglobin is encapsulated and then exposed to CO, the CO rebinding kinetics are characteristic of the initial low affinity T quaternary structure associated with deoxy hemoglobin. Similarly, when CO saturated hemoglobin is encapsulated the resulting kinetics are characteristic of the high affinity R structure without any evidence for the usual R to T transition that occurs subsequent to photodissociation for solution phase samples. The locking in of quaternary structures is supported by several spectroscopic findings.