Investigating Ribonuclease A Modification Induced by Quinones Using UV/Vis Spectroscopy

Transcription

1 Investigating Ribonuclease A Modification Induced by Quinones Using UV/Vis Spectroscopy Caitlin Redman, Albert Vaughn, Steve Ledford and Jisook Kim, University of Tennessee at Chattanooga, Chattanooga, TN

2 Introduction The goal of this research project is to have a better understanding on the biochemical behavior and biological effect(s) of various quinones that are known to be cellular metabolites of benzene and substituted benzenes. Benzene as well as polycyclic aromatic hydrocarbons are considered to be health hazards. Exposure to these chemicals can occur in a variety of ways such as cigarette smoke, gasoline vapors, pesticides, cosmetics, and food preservatives. This research project primarily focuses on studying the effects that certain quinones have on proteins. Ribonuclease A, RNase, is the model protein used for this project. Several studies suggest that quinones can cause modifications that lead to toxic and abnormal cell behavior. Such modifications include oxidative damage, lipid modification, and/or nucleic acid modifications. There are, however, many details concerning the outcome of such interactions which are not clearly understood. The interaction and subsequent modification of RNase with three different quinones,,-benzoquinone, -chloro-,- benzoquinone, and -methyl-,-benzoquinone, are studied using a UV/Vis spectroscopic approach.

3 Quinones Studied O O O O Cl O O CH -chloro-,-benzoquinone (ClpBQ),-benzoquinone (pbq) -methyl-,-benzoquinone (MepBQ) Polycyclic aromatic hydrocarbons (PAHs) are wide spread environmental toxins. Exposure to these chemicals can occur in a variety of ways. Such ways include: Cigarette Smoke Grilled Meat Paint & Furniture Polish Petroleum Products Food Preservatives The three quinones shown on the left were used during this research project.

4 Proposed Mechanism Oxidative damage Pathway I O O - OH OH [O] O O Protein Nu Pathway II Adduct formation H N O Lys + H N Lys Lys Pathway III Protein crosslink

5 Sequence Map of Ribonuclease A Smyth, D.G., Stein, W.H., and Moore, S. J. Biol. Chem. 8, 7 (96) KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKP VNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCR ETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

6 The Binding of IMP to Ribonuclease A Febs J. v7 pp. 988-, 5 (Z6D)

7 Time Dependent UV/Vis Spectra of RNase Control This graph represents the time dependent stability test of.5mg RNase in a 5mM phosphate buffer, ph 7. The spectrum was scanned every minutes for hours. The spectral data shown demonstrates that our model protein, RNase A, is a stable molecule..5mg RNase in phosphate buffer, ph nm

8 Time Dependent Stability Test of Quinones The time dependent stability of our quinones were studied by taking a spectral scan on the UV/Vis every hour for hours using. The quinones were in a 5mM phosphate buffer, ph 7 and the temperature was set at 7 C. 5 mm Methyl-pBQ Stability Test 5 nm mm p BQ Stability Test 5 mm Cl-p BQ Stability Test 9 nm 9 nm

9 UV/Vis Reactions of pbq and RNase at Various [pbq] mm pbq,.5mg RNase Initial Scan mm pbq,.5mg RNase Initial Scan mm pbq,.5mg RNase Initial Scan mM pbq,.5mg RNase Initial Scan

10 Representative Spectra of RNase Modification by pbq A bsorbance mm pbq,.5mg RNase.5mM pbq and.5mg RNase A bsorbance Initial Scan Spectral Scan every mins for hours Spectral Overlay Daily Spectral Scans for approximately weeks.

11 Determining the Rate Constant Time Dependent Progress of RNase Modification by pbq Time (hours).5mm pbq mm pbq mm pbq mm pbq 5mM pbq Time Dependent Progress of RNase Modification by pbq at 5nm.5.5 Time (hours).5mm pbq mm pbq mm pbq mm pbq 5mM pbq The rate constant of RNase modification by pbq was calculated by selecting a chromophore that was unique to the spectrum of RNase plus quinone. In the case of pbq, a wavelength of 5nm was selected and plotted against time (hrs). Application of the appropriate equations give rise to the reaction rate constant.

12 Kinetic Analysis Rate constant (hr-) Kinetic model: Pseudo first order condition [pbq] is in excess relative to RNase A. U P Kinetic equation used: ln A -A t A -A = -k t Rate Constants: Reaction of RNase A & pbq 5 6 [pbq] (mm) U: Unmodified RNase P: Modified RNase A : of P at the time infinity A t : of P at time t A : Initial absorbance of P k : Pseudo first order rate constant t : Reaction time

13 UV/Vis Scans of mm Methyl-pBQ & mm Cl-pBQ A bsorbance mm Methyl-pBQ,.5mg RNase Initial Scan mm Cl-pBQ,.5mg RNase Initial Scan The rate constant for the Methyl-pBQ and Cl-pBQ has not yet been determined. The spectral data generated for these two quinones did not possess a chromophore unique only to the reaction between the quinone and RNase. Further experimentation will be completed in order to calculate the rate constant.

14 Finding the Optimal Conditions for Dialysis 5 mm p-bq 6hr Dialysis (Tubing) 5 mm p-bq 6hr Dialysis (Snake Skin) Before Dialysis After Dialysis Before Dialysis After Dialysis Wavlength (nm) 5 mm p-bq hr Dialysis (Tubing) 5 mm p-bq hr Dialysis (Tubing) Before Dialysis After Dialysis Before Dialysis After Dialysis Wavlength (nm)

15 UV/Vis Data After Removal of Excess Quinones mm p-bq,.5mg RNase After hr Dialysis hr Incubation Time hr Incubation Time 9hr Incubation Time 7hr Incubation Time mm Cl-pBQ,.5mg RNase After hr Dialysis hr Incubation Time hr Incubation Time 9hr Incubation Time 7.5hr Incubation Time

16 DNP Trapping Experiment A. Intact protein B. Modified protein C. DNP tagged protein Quinone,-DNP O N Dialysis Spectral Scan Lys NH Lys O Lys N NH NO Unmodified RNase treated with,-dnp A bsorbance Modified RNase treated with,-dnp

17 References Bolten, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., Monks, T. J. () Role of Quinones in Toxicology. Chem. Res. Toxicol., 5-6. Bolten, J. L., Pisha, E., Zhang, F., and Qiu, S. (998) Role of quinoids in estrogen carcinogensis. Chem. Res. Toxicol., -7. Monks, T. J., Hanzlik, R. P., Cohen, G. M., Ross, D., and Graham, D. G. (99) Contemporary issues in toxicology: Quinone chemistry and toxicity. Toxicol. Appl. Pharmacol., -6. O Brien, P. J. (99) Molecular mechanisms of quinone cytotoxicity. Chem.-Biol. Interact. 8, -.