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 hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the DeltaD vs. Deltaf graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated 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. Thus, 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 about the mechanisms of protein adsorption to solid surfaces.
Höök
F
F
Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, Medicinaregatan 9C, SE-413 90 Göteborg, Sweden. fredrik.hook@bcbp.gu.se
Rodahl
M
M
Kasemo
B
B
Brzezinski
P
P
D000327:Adsorption; D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry; D002417:Cattle; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation
D006454:Hemoglobins; D008024:Ligands; D002248:Carbon Monoxide