Fluorescence lifetime measurements using total internal reflection fluorimetry: evidence for a conformational change in albumin adsorbed to quartz.
We have modified the total internal reflectance fluorimetry (TIRF) method to determine, indirectly, quantum fluorescence yield and thus infer structural changes of adsorbed plasma proteins. We employed a frequency multiplied Nd:YAG pulse laser to excite tryptophan residues of bovine serum albumin (BSA), measuring the resulting fluorescence lifetimes. BSA adsorbed on quartz was investigated at normal pH and under acidic (pH 3.8) conditions at which albumin shape change has been established in bulk solution. Albumin adsorbed at pH 7.2 has a slightly lower lifetime than the bulk solution value, reflecting a population of both native and isomerized forms. Gently washing the adsorbate surface significantly reduces fluorescence lifetime, suggesting retention of more tenacious, conformationally altered molecules. Vigorous washing further reduces fluorescence lifetimes to values close to those obtained in bulk under acidic conditions. Similar alterations in fluorescence lifetimes were observed for heat-denatured albumin. These results support the hypothesis that adsorbed albumin exists in the form of a two-layer deposit, each with a possibly different structure. A loosely held layer, consisting of BSA microaggregates and including partially unfolded molecules, exists on the undisturbed surface and is partially removed by gentle washing. Vigorous washing removes all but the most tightly held protein, which exists in a possibly further unfolded configuration. The decrease in fluorescence lifetime of adsorbed BSA implies a corresponding change in quantum yield. A change in quantum yield of BSA tryptophan fluorophors implies that measuring adsorbed protein concentration by intrinsic fluorescence is unreliable.
D000327:Adsorption; D005453:Fluorescence; D005470:Fluorometry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011791:Quartz; D012710:Serum Albumin, Bovine / Q000032:analysis; D014364:Tryptophan / Q000032:analysis
D011791:Quartz; D012710:Serum Albumin, Bovine; D014364:Tryptophan