Quenching of beta-lactoglobulin fluorescence by 2-nitro-5-thiobenzoic acid.

I3C-NMR studies of modified 6-lactoglobulins, cyanylated at the single thiol group, have provided evidence to support the location of the thiol at Cys-121 [ I J and useful information about the nuclear relaxation behaviour of the thiocyanate carbons of cyanylated thiol groups in proteins [2]. The O-lactoglobulin monomer contains two tryptophan residues. Trp-19 is buried deep inside the hydrophobic 6-barrel core of the protein and Trp61 is located in a flexible loop. The thiol group of Cys-121 is located in a hydrophobic cleft near the surface of the protein [3]. The binding of retinol by 6-lactoglobulin significantly quenches the inbinsic fluorescence of the protein [4]. Trp-19 is the only invariant residue in the lipocalin superfamily, and it has been implicated in the stabilisation of retinol at it's binding site, although it is not essential for retinol binding [51. The intrinsic fluorescence of 6-lactoglobulin is mainly due to Trp-19, since the fluorescence of a W19A mutant protein is only 20 % of that of wild-type protein [6]. The mixed disulphide derivative of 0-lactoglobulin B with 2-nitro-5-thiobenzoic acid (TNB) was prepared using 5,5'dithiobis(2,T-nitrobenzoic acid) (DTNB) as previously described 111. Fluorescence measurements were made at 25 O C using a Hitachi Model F-4500 fluorimeter. Fluorescence emission spectra of 8-lactoglobulin B and the mixed disulphide derivative were recorded using an excitation wavelength of 290 nm and excitation and emission slit widths were fixed at 4 nm. 1 ml 8lactoglobulin solutions used for fluonmetry contained 6-10 pM monomer, in 10 mM potassium phosphate buffer at pH 7.0. Distances between amino acid side-chains in 6-lactoglobulin were measured using SwissPdbViewer version 2.1 from the Glaxo Institute for Molecular Biology, Geneva, Switzerland. Coordinates for bovine 0-lactoglobulin were kindly supplied by L. Sawyer, Structural Biochemistry Group, University of Edinburgh. The intrinsic tryptophan fluorescence of 8-lactoglobulin B has a maximum emission wavelength at 335 nm (Fig. IA), indicative of a hydrophobic environment around the tryptophan residues.