Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence.
The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermal quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, 1/q, vs. the T/eta ratio (T, temperature; eta, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva , E.P. Busel and E.A. Burstein , Biochim. Biophys, Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg2+-loaded whiting parvalbumin (tryptophan fluorescence), Mg2+-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca2+-loaded bovine alpha-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.
D000818:Animals; D002118:Calcium; D002152:Calorimetry, Differential Scanning; D005399:Fishes; D008274:Magnesium; D009124:Muscle Proteins; D009132:Muscles; D010320:Parvalbumins; D010649:Phenylalanine; D011487:Protein Conformation; D013045:Species Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D014364:Tryptophan; D014443:Tyrosine
D009124:Muscle Proteins; D010320:Parvalbumins; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium