1. Correlation between dynamics, structure and spectral properties of human  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  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 orosomucoid c –2) Ammonium sulfate precipitation Denoted as orosomucoid a

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= (I v -gI h )/(I v + 2gI h ) Time decay of fluorescence intensity –Edinburgh Analytical Instruments CD 900 fluorometer

10. Results Lifetime data –Orosomucoid a Fluorescence intensity of Trp residues ex =295 nm, em =330 nm I(,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 –Orosomucoid c Decay times 0.35, 1.66, and 4.64  0 = 2.23 ns

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

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

13. Excitation anisotropy spectra a=orosomucoid a b=orosomucoid b em =330 nm at 20°C

14. Anisotropy as a function of emission a=orosomucoid a b=orosomucoid b ex =295 nm at 20°C

15. Conclusion Trp residues of orosomucoid b display free motion and those of orosomucoid a 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 ex and em arise from: –Structure and dynamics of the microenvironment of Trp –Tertiary structure of the protein