THE EFFECT OF HEAT-TREATMENT ON ANILINONAPHTHALENE-8-SULFONATE BINDING TO RAPESEED ALBUMIN (NAPIN)

Protein surface hydrophobicity can be measured by the fluorescent prob e method. The effect of heat treatment on Brassica napus (rapeseed) al bumin (napin) interactions with a fluorescent probe, anilinonaphthalen e-8-sulphonic acid (ANS) was investigated by fluorescent titration. Up on heating to 100 degrees C for 30 min the number of napin binding sit es for ANS (n) increased from 5(+/- 0.7) to 13(+/- 0.5) moles of ANS b ound per mole of protein. The ANS-protein dissociation constant (Kd) w as 2.0(+/- 0.4) x 10(-6) M for the unheated protein and 10.4(+/- 0.1) x 10(-6) M for heat-denatured napin. There was also a blue shift in th e fluorescence emission spectrum maximum for denatured napin-ANS compl ex consistent with an increase in the hydrophobicity of the ANS bindin g sites in the denatured protein. The characteristic fluorescence incr ease for heat-denatured albumin-ANS mixtures is therefore due to an in crease in the number, binding affinity and hydrophobicity of binding s ites. Heat treatment of napin leads to the appearance of additional su rface hydrophobic sites in the denatured protein.