Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding

Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding

^*Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, Medicinaregatan 9C, SE-413 90 Göteborg, Sweden; and ^†Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden

Communicated by Hans Frauenfelder, Los Alamos National Laboratory, Los Alamos, NM (received for review November 26, 1997)

Abstract

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 ΔD vs. Δf 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.

Footnotes

↵ ‡ To whom reprint requests should be addressed. e-mail: fredrik.hook{at}bcbp.gu.se or peter{at}bcbp.gu.se.
↵ § Because met-Hb may denature at room temperature after ≈1 hr (23), all experiments were interrupted after ≈2,000 s. Since ∂f/∂t was non-zero at this time, the adsorption process was still in progress. For the present comparative purposes this definition of asymptotic values is, however, sufficient.
↵ ¶ The change in f is related to the effective mass (16). However, when relative variations in the f-shifts are discussed, the absolute value is of minor importance.
↵ ‖ It should be noted that the amplitude of oscillation is typically ≈1 nm. This means that the crystal surface moves with a velocity < 1 cm/s and that the maximum kinetic energy transferred to the proteins due to the oscillation is much smaller than k _BT. The oscillatory motion of the crystal is therefore not expected to significantly influence the structure or adsorption behavior of the protein molecules.
ABBREVIATIONS:

QCM,

Quartz Crystal Microbalance;

f,

frequency;

D,

energy dissipation;

pI,

isoelectric point