Fluorescence and circular dichroism spectroscopic studies on bovine lactoperoxidase*

Abstract

Intrinsic steady-state fluorescence of lactoperoxidase (LPO) and its ligand-bound complexes has been characterized as a structural probe of its structure in solution. On excitation at 295 nm, a broad emission maximum is observed around 338 nm for LPO and for its ligand-bound complexes. The quantum yield is 0.0185±0.0005 for LPO and indicates tryptophan → heme energy transfer. Tryptophan residues are located away from heme and are approximately equally distributed among hydrophobic and hydrophilic environments. From Förster resonance energy transfer equations, the “average” distance between tryptophans and heme within the enzyme is computed to be 25.1±0.2 Å. These fluorescence properties are consistent with the recent theoretical three-dimensional model for LPO and reveal that Trp337 and Trp404 dominate the intrinsic fluorescence, and together contribute ∼ 64% of the observed intensity. The effects of the denaturing agents guanidine hydrochloride and urea on the intrinsic fluorescence of LPO and CD of the backbone amide chromophores have been examined. The considerably red shifted emission maximum at 356 nm indicates that tryptophans, buried in the hydrophobic environment, are exposed to the solvent on denaturation. A simple two-state transition between the native and denatured forms of the protein has been used to explain the results. [Denaturant]_1/2∼ 5.5 M, determined from both these experiments, indicates that LPO is relatively stable toward the denaturing agents. Quenching studies using. I⁻, Cs⁺ and polar neutral acrylamide are consistent with this picture. Acrylamide can penetrate the protein matrix. It is an efficient quencher and the quenching process is essentially homogeneous with all the tryptophans being accessible. Cs⁺ ion is a very inefficient quencher but the iodide ion shows the quenching process to be predominantly heterogeneous with widely differing tryptophan accessibility. The Stern–Volmer constants deduced are K ^sv=8.4±1.4 M⁻¹ and K ^sv=4.05±0.65 M₋₁ for acrylamide and iodide quenching, respectively. The fractional accessibility, f _a , deduced is f _a =0.52±0.03 for iodide quenching.