SPECTROSCOPIC PROPERTIES AND STABILITY OF HEMOCYANINS

The stability towards thermal and chemical (guanidine hydrochloride) d enaturation of oxy- and apo-hemocyanins from the arthropodan organisms Homarus americanus, Maia squinado, Palimurus vulgaris and Carcinus ma enas as well as from the molluscs Rapana thomasiana and Viviparus ater have been investigated by fluorescence spectroscopy and circular dich roism. The H. americanus hemocyanin showed an extreme thermostability in comparison to the other investigated hemocyanins. The critical temp erature 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 dea ctivation 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 dichr oism. 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 m ol(-1). The highest Delta G(D)(H2O)-values were calculated for the Rap ana thomasiana hemocyanin. Upon excitation at 295 or 280 nm the fluore scence emission of the investigated hemocyanins is dominated by 'burie d' tryptophyl chromophores. The removal of the copper-dioxygen system from the active site led to 3.8-7.9-fold increase of the protein fluor escence quantum yield and to a red shift of the emission maximum posit ion. Evidently, the tryptophyl fluorescence is significantly quenched in the oxy-hemocyanins. (C) 1997 Elsevier Science B.V.