Solvent effect on librational dynamics of spin-labelled haemoglobin by ED- and CW-EPR.
Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine β93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude-correlation time product, <α²>τ(c), deduced from the ED-EPR spectra. The librational amplitude, <α²>τ(c), was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ(c), was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, <α²> deviated from small values only for T > 200 K, where a rapid increase of <α²> was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.
Dipartimento di Fisica, Laboratorio di Biofisica Molecolare and UdR CNISM, Università della Calabria, 87036 Arcavacata di Rende, CS, Italy.
Francesco
F
Scarpelli
Rosa
R
Bartucci
Luigi
L
Sportelli
Rita
R
Guzzi
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008956:Models, Chemical; D009038:Motion; D012997:Solvents / Q000737:chemistry*; D013113:Spin Labels / Q000138:chemical synthesis*; D013696:Temperature
D006454:Hemoglobins; D012997:Solvents; D013113:Spin Labels