T- and R-state Tertiary Relaxations in Sol-gel Encapsulated Haemoglobin
Tertiary relaxations within the T and R quaternary states of human adult haemoglobin (HbA) are compared for sol-gel encapsulated samples bathed in buffer with either 25% or 75% (v/v) glycerol. T-state tertiary relaxations are initiated by adding CO to an encapsulated T-state deoxyHbA sample, thus generating liganded T-state species. The conformational evolution of the liganded T-state samples is followed by monitoring the frequency of v(Fe-His), the conformation-sensitive iron-proximal histidine stretching mode observed in the resonance Raman spectra of either of the deoxy sample of the 7 ns photoproduct derived from the CO samples. In parallel, the functional properties are monitored by following the evolution of the kinetic traces associated with CO recombination subsequent to nanosecond photodissociation of the CO-heme unit. In contrast, the R-state relaxations are initiated by adding dithionite to encapsulated samples of either oxyHbA or cyanometHbA, thus generating deoxy hemes whose resonance Raman spectra reflect the influence of the relaxing tertiary structure within the R state. After the “deoxy” sample is allowed to relax for a defined time period, CO is introduced. The evolution of the relegated samples is now followed by monitoring the photoproduct frequency of the v(Fe-His) Raman band and the kinetic traces for the CO recombination.
The results reveal a hierarchy of R/T-dependent tertiary relaxation processes whose differences can be explained based on differences in solvent slaving properties of the different relaxations. The results also support models of HbA allostery in which there are multiple functionally distinct tertiary conformations with each quaternary state.
KeywordsEnergy Landscape Kinetic Trace Resonance Raman Spectrum Human Hemoglobin Allosteric Effector
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