Exposure of tryptophanyl residues in?-lactalbumin and lysozyme

Abstract

The effect of iodide ion on the tryptophyl fluorescence of the homologous proteins lysozyme and alpha-lactalbumin in their native form, as well as in their modified structures and in fragments from these proteins was studied. By assessing the contribution to the total fluorescence of the exposed and buried Trp residues, and of the respective fluorescence quantum yields, the quantification of the number of Trp exposed to the solvent for all the species studied was possible. Both native proteins show an important increase in the number of Trp residues exposed to the solvent when treated with denaturing agents. The peptides L-II (aa 13-105) and alpha-I (aa 1-90) from lysozyme and alpha-lactalbumin, respectively, showed Trp residues with different degree of exposure, whereas the smaller fragments, L-III (aa 106-129) and alpha-II (aa 91-123), had all their Trp residues exposed to the solvent.

Poly(carboxybetaine methacrylamide)-Modified Nanoparticles: A Model System for Studying the Effect of Chain Chemistry on Film Properties, Adsorbed Protein Conformation, and Clot Formation Kinetics

“…In comparing peak maxima, it is evident that a red shift occurs upon adsorption to PCBMA samples, being most prominent for SiNP-PCBMA5 with TEMPO end-groups (∼338 nm), perhaps indicating that the protein matrix changed such that tryptophan residues are in a more solvated local environmentthe effect of monomer being considered to be greater than the end-group in altering this protein conformation, from this measurement. The 3D structure of α-la has previously been reported, and it is known that the four tryptophan residues are generally located in the internals of the protein, away from solvent . Hence, a red shift would most likely indicate a loosening/unfolding of the protein induced by the adsorption onto various SiNPs.…”

“…Acrylamide was employed as it can penetrate into the protein matrix and quench the buried fluorophore. If the protein has both accessible and inaccessible fluorophores, the Stern–Volmer (Lehr) relationship (eq ) can be used to quantify the efficiency of quenching and the accessibility of tryptophan residues, before and after adsorption. − I 0 I 0 − I = 1 f normala + 1 f normala K sv false[ normalQ false] where I 0 is the unquenched intensity, I is the quenched intensity, f a is the fraction of quenched fluorophore, K sv is the Stern–Volmer constant (M –1 ), and [Q] is the concentration of quencher (M). The comparison of K sv and f a of native and adsorbed protein should further provide details into the changes in local environment of tryptophan residues.…”

“…Peak intensities were read directly from the corrected emission spectra and analyzed using a modified Stern–Volmer (Lehr) plot. − …”

“…The 3D structure of α-la has previously been reported, and it is known that the four tryptophan residues are generally located in the internals of the protein, away from solvent. 66 Hence, a red shift would most likely indicate a loosening/unfolding of the protein induced by the adsorption onto various SiNPs.…”

Fluorescence Quenching Reactions

“…In general, alterations may include (1) conformational changes or aggregation/disaggregation induced by temperature, ionic strength, pH, denaturants, and so on and (2) conformational changes induced by the binding of specific ligands (see Eftink, 1991, for a list of example studies). Or comparison may be made of (3) homologous biomacromolecules (Edwards and Silva, 1986;Sommers and Kronman, 1980), including proteins made by site-directed mutagenesis (Hansen et a/., 1987).…”