Spectroscopic properties and stability of hemocyanins

The stability towards thermal and chemical (guanidine hydrochloride) denaturation of oxy- and apo-hemocyanins from the arthropodan organisms Homarus americanus, Maia squinado, Palimurus vulgaris and Carcinus maenas as well as from the molluscs Rapana thomasiana and Viviparus ater have been investigated by fluorescence spectroscopy and circular dichroism. The H. americanus hemocyanin showed an extreme thermostability in comparison to the other investigated hemocyanins. The critical temperature of deviation from linearity (T-c) of the Arrhenius plot, ln(Q(-1) - 1) vs. 1/T, where Q is the protein quantum yield of fluorescence, was calculated to be 87 degrees C for this respiratory protein. The T-c-values for the other hemocyanins range between 63 and 76 degrees C. The respective activation energies for the radiationless thermal deactivation of the excited indole chromophores were calculated to be 37.0-50.5 kJ mol(-1). Guanidine hydrochloride is an efficient denaturant for hemocyanins. The protein unfolding was monitored by circular dichroism. The foe energy of stabilization in water, Delta G(D)(H2O), at 25 degrees C and pH 7.5, was calculated to be in the range 8.0-21.6 kJ mol(-1). The highest Delta G(D)(H2O)-values were calculated for the Rapana thomasiana hemocyanin. Upon excitation at 295 or 280 nm the fluorescence emission of the investigated hemocyanins is dominated by 'buried' tryptophyl chromophores. The removal of the copper-dioxygen system from the active site led to 3.8-7.9-fold increase of the protein fluorescence quantum yield and to a red shift of the emission maximum position. Evidently, the tryptophyl fluorescence is significantly quenched in the oxy-hemocyanins. (C) 1997 Elsevier Science B.V.