Protein adsorption at the solid/liquid interface monitored by electron spin resonance spectroscopy

Abstract Electron spin resonance (ESR) was used to determine the amount of bovine serum albumin (BSA) adsorbed at a solid/liquid interface and to monitor the conformational changes of the protein as a consequence of the adsorption. The technique depends on allowing a spin-labelled protein in solution to flow through a column placed inside the resonance cavity of the electron spin resonance spectrometer. BSA was spin labelled with 3-(maleimidomethyl)-proxyl (MSL) free radical to yield spin-labelled BSA (BSA—MSL). BSA—MSL gives a broad asymmetric ESR spectrum with a maximum hyperfine splitting 2 T II = 64.1 G at 22°C and s/w = 0.45, where s/w , a semiquantitative parameter, is the ratio of the amplitudes of the low magnetic field peaks. The parameters indicate the immobilization of the MSL covalently bound to the BSA molecule. The ESR spectra of BSA—MSL adsorbed on to the surface of controlled pore glass beads show additional restrictions to the MSL motion as indicated by 2 T II = 66.5 G and s/w = 0.82. The results suggest that the site of the protein adsorption on to the solid surface is not in the immediate vicinity of the cysteine residue to which MSL is covalently bound. ESR spectra of fatty-acid-free BSA—MSL and BSA—MSL treated with 10% sodium dodecyl sulfate show a considerable flexibility in the movement of the nitroxide moiety which is not additionally restricted when the spin-labelled protein molecule is adsorbed on to the glass surface. The amount of MSL—BSA adsorbed on the glass surface was determined to be 4.2 μg m −2 .