J.R.Albani Susan Ahrens April 13, 2004

1. Correlation between dynamics, structure and spectral properties of human a 1-acid glycoprotein (orosomucoid): a fluorscence approach. J.R.Albani Susan Ahrens April 13, 2004

2. Outline • Introduction • Aims of the study • Methods • Results • Conclusions

3. Introduction • Human a 1 acid-glycoprotein- Orosomucoid • Plasma glycoprotein • MW=41 kDa (181 amino acids) • 40% carbohydrate (by weight) • pI of 2.8-3.4 • Functions • Little known • Binds to several compounds • Warfarin, vanilloids, IgG, heparin and steroid hormones

4. Previous Work • Three tryptophan residues • Environment of the rotating unit • Use of red-edge excitation spectra • Dynamics of proteins and membranes • Motion of fluorophore compared to motion of macromolecule • Flexibility of microenvironment

5. Aims of present study • Microenvironment of Trp • Red-edge excitation spectra and anisotropy • Applied to Trp of orosomucoid from two preparations • Chromatographic methods • Ammonium sulfate precipitation • Anisotropy as a function of excitation and emission wavelengths

6. Terms • Red shift excitation shift • Results when a polar fluorophore is under conditions where solvent relaxation is not complete. • Emission spectra shifts to long wavelengths when excitation is on the long wavelength edge of the absorption spectrum. • Anisotropy • Describes the extent to which emission light is polarized. • Depends on the transition moments for absorption and emission which lie along certain directions within a fluorophore.

7. Methods • Two sources of orosomucoid: • 1) Combination of ion displacement chromatography, gel filtration, and ion exchange chromatography • Denoted as orosomucoidc • 2) Ammonium sulfate precipitation • Denoted as orosomucoida

8. Methods • Fluorescence spectra • Recorded with a Perkin- Elmer LS-5B spectrofluorometer at a bandwidth of 2.5 nm for excitation and emission • Optical path lengths 1 cm for emission and 0.4 cm for excitation • Intensities were corrected for • 1) Dilution • 2) Absorption • 3) Background of buffer

9. Methods • Perrin plot • Fluoroscence anisotropy A= (Iv-gIh)/(Iv + 2gIh) • Time decay of fluorescence intensity • Edinburgh Analytical Instruments CD 900 fluorometer

10. Results • Lifetime data • Orosomucoida • Fluorescence intensity of Trp residues lex=295 nm, lem=330 nm I(l,t) = 0.455 e-t/0.25 + 0.496 e-t/1.75 + 0.049 e-t/5.15 • Mean fluorescence lifetime 0 = 2.29 ns • Orosomucoidc • Decay times 0.35, 1.66, and 4.64 0 = 2.23 ns

11. Red-edge excitation spectra Fluorescence spectra for orosomucoida lex = a- 295 nm, b- 300 nm, c- 305 nm

12. Fluorescence anisotropy as a function of temperature Data for orosomucoida thermal range 7-35°C. lex=300 nm, lem=330 nm Perrin plot

13. Excitation anisotropy spectra a=orosomucoida b=orosomucoidb lem=330 nm at 20°C

14. Anisotropy as a function of emission a=orosomucoida b=orosomucoidb lex=295 nm at 20°C

15. Conclusion • Trp residues of orosomucoidb display free motion and those of orosomucoida are rigid. • As the motions of the fluorophore are constrained by the microenvironment, larger decreases in the anisotropy along the emission wavelength are seen. • Dependence of anisotropy on lex and lem arise from: • Structure and dynamics of the microenvironment of Trp • Tertiary structure of the protein