Resolution of the heterogeneous fluorescence in multi-tryptophan proteins: ascorbate oxidase.
Ascorbate oxidase is a copper-containing enzyme which catalyzes a redox reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Rosato, N. & Finazzi Agrò, A. (1997) Biochemistry 36, 10917-10922]. In this study, the steady-state and dynamic fluorescence features of ascorbate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity and anisotropy upon excitation at 295 nm are extremely sensitive functions of the emission wavelength, indicating a great heterogeneity of the system. The emission decay collected through a cut-off filter can be analyzed in terms of two continuous distributions of lifetimes. Using a monochromator in emission or an optical multichannel analyzer, the two distributions may be attributed to distinct components of the fluorescence spectrum. Differential quenching by cesium chloride also confirmed that the several tryptophan residues present in the protein structure may be grouped into two main classes, each with a different environment. Once the complex fluorescence decay of ascorbate oxidase was analyzed and resolved, a comparison with the crystallographic data allowed a first, approximate attribution of the protein spectroscopic properties to some of the tryptophan residues. This might provide a powerful tool of investigation about the role of definite segments of the protein in its three-dimensional structure and catalytic activity. Furthermore, the methodology set up for ascorbate oxidase can be usefully extended to other multitryptophan proteins.
Di Venere
A
A
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
Mei
G
G
Gilardi
G
G
Rosato
N
N
De Matteis
F
F
McKay
R
R
Gratton
E
E
Finazzi Agrò
A
A
D001204:Ascorbate Oxidase / Q000737:chemistry*; D005454:Fluorescence Polarization; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry*; D014675:Vegetables / Q000201:enzymology
D014364:Tryptophan; D001204:Ascorbate Oxidase