The Effect of Heat Treatment on Anilinonaphthalene‐8‐Sulphonate Binding to Rapeseed Albumin (Napin)

Protein surface hydrophobicity can be measured by the fluorescent probe method. The effect of heat treatment on Brassica napus (rapeseed) albumin (napin) interactions with a fluorescent probe, anilinonaphthalene-8-sulphonic acid (ANS) was investigated by fluorescent titration. Upon heating to 100°C for 30 min the number of napin binding sites for ANS (n) increased from 5(±0.7) to 13(±0.5) moles of ANS bound per mole of protein. The ANS-protein dissociation constant (Kd) was 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 the fluorescence emission spectrum maximum for denatured napin-ANS complex consistent with an increase in the hydrophobicity of the ANS binding sites in the denatured protein. The characteristic fluorescence increase for heat-denatured albumin-ANS mixtures is therefore due to an increase in the number, binding affinity and hydrophobicity of binding sites. Heat treatment of napin leads to the appearance of additional surface hydrophobic sites in the denatured protein.