STRUCTURAL-CHANGES IN HEMOGLOBIN DURING ADSORPTION TO SOLID-SURFACES - EFFECTS OF PH, IONIC-STRENGTH, AND LIGAND-BINDING

We have studied the adsorption of two structurally similar forms of he moglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-term inated thiol monolayer on a gold surface, by using a Quartz Crystal Mi crobalance (QCM) technique. This technique allows time-resolved simult aneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM du ring the adsorption process, which makes it possible to investigate th e viscoelastic properties of the different protein layers during the a dsorption process. Below the isoelectric points of both met-Hb and HbC O, the Delta D vs. Delta f graphs displayed two phases with significan tly different slopes, which indicates two states of the adsorbed prote ins with different visco-elastic properties. The slope of the first ph ase was smaller than that of the second phase, which indicates that th e first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was assoc iated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe3+ of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Th us, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both D and f, demonstrating that the QCM technique provides unique information a bout the mechanisms of protein adsorption to solid surfaces.