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Determining the Role of Proteins in the Molecular Properties of Equine Synovial Fluid

Determining the Role of Proteins in the Molecular Properties of Equine Synovial Fluid. Marsha Lampi Advisor: Dr. Skip Rochefort Oregon State University School of Chemical, Biological and Environmental Engineering Summer 2009. Synovial Fluid. Found in diarthrotic , freely moveable joints.

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Determining the Role of Proteins in the Molecular Properties of Equine Synovial Fluid

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  1. Determining the Role of Proteins in the Molecular Properties of Equine Synovial Fluid Marsha Lampi Advisor: Dr. Skip Rochefort Oregon State University School of Chemical, Biological and Environmental Engineering Summer 2009

  2. Synovial Fluid • Found in diarthrotic, freely moveable joints. • Responsible for nutrient distribution, lubrication, and shock absorption. • Used for diagnosis of joint diseases. Synovial Membrane Synovial Joint Cavity Articular Cartilage Articulating Bone http://edugen.wiley.com/edugen/student/mainfr.uni

  3. Hyaluronic Acid (HA) • Largest molecular component of synovial fluid. Molecular weight ranges from 0.2 – 10 million g/mol. • Some joint diseases have been linked to the breakdown of HA. • HA injections and oral supplements are currently available and being studied as treatments for joint diseases.

  4. Proteins • Plasma proteins: albumin and globulin • Molecular weight range of 40 – 60 thousand g/mol. http://www.scielo.br/img/revistas/bjmbr/v42n4/html/7566i01.htm

  5. Equine Synovial Fluid Stifle (knee) Hock (ankle) http://www.ucmp.berkeley.edu/education/lessons/xenosmilus/skeletal_res_manual2.html

  6. Objective Develop a protocol to digest the protein in synovial fluid while leaving the hyaluronic acid unchanged.

  7. Methodology • Remove the protein through protease digestion. • Analyze the molecular composition of synovial fluid with light scattering. • Analyze the molecular composition of the digested synovial fluid to verify the protein had been eliminated.

  8. Analysis of Molecular Composition

  9. Gel Permeation Chromatography(GPC) • Separates particles based on size. • Small particles get stuck in the packed interior and move through the column slower. http://www.ap-lab.com/images/LS_setup.gif http://www.waters.com/waters/partDetail.htm ?locale=en_US&partNumber=WAT045915

  10. Polymer Solution Light Source  Detector, Io Detector, I() Multi-Angle Laser Light Scatter(MALLS) • Light intensity is measured as a function of the deflection angle and concentration. • Allows for molecular weight determination.

  11. Refractive Index (RI) Detector • Determines concentration based on the bending of light in comparison to a reference cell. http://www.polygen.com.pl/viscotek/refractive_index_detector.html

  12. Experimental Set-Up http://www.ap-lab.com/images/LS_setup.gif

  13. GPC-MALLS Graph -- Light Scatter -- Refractive Index Protein Peak HA Peak Before digestion, both the HA and protein peaks are detected.

  14. Protein Digestion • ProteaseBacillus polymyxa, 1.2 U/mg • Preliminary digestion: • Dilute synovial fluid sample 1:3 • 2 units of protease per mL synovial fluid • 30 minute incubation in water bath • Filtration and phenol-chloroform extraction to remove proteins Kvam, Catrine, Granese Daniela, Flaibani, Antonella, Zanetti, Flavio, and Paoletti, Sergio (1993). “Purification and Characterization of Hyaluronan form Synovial Fluid”. Analytical Biochemistry 1993, 211, 44-49.

  15. Undigested Synovial Fluid -- Light Scatter --Refractive Index Protein Molecular Weight: 6-8 x 104 g/mol Digested Synovial Fluid -- Light Scatter --Refractive Index Protein Molecular Weight: 3-6 x 103 g/mol

  16. Protease Concentration • Increase to 4 units/mL Synovial Fluid • Same elution time = no gain in digestion • Low HA concentration -- Light Scatter --Refractive Index Protein Molecular Weight: 3-6 x 103 g/mol

  17. Removal of Dilution Step • 2 units protease/mL Synovial Fluid • Same elution time = no gain in digestion • Low HA concentration continues. Possibly removed in filtration step. -- Light Scatter --Refractive Index Protein Molecular Weight: 3-6 x 103 g/mol

  18. Removal of Filtration Step • 2 units protease/mL Synovial Fluid • Same elution time = no gain in digestion • Low HA concentration continues. Possibly removed in phenol-chloroform extraction. -- Light Scatter --Refractive Index Protein Molecular Weight: 3-6 x 103 g/mol

  19. Addition of Protease Only • 2 units protease/mL Synovial Fluid • Earlier elution time = Less digestion • There is no change between the digested and undigested samples when treated with the protease. -- Light Scatter --Refractive Index Protein Molecular Weight: 5-7 x 104 g/mol

  20. Conclusion • The proteaseBacillus polymyxa currently being used is not effective in digesting the equine synovial fluid proteins. The protein removal may only be due to phenol-chloroform extraction. • Protein digestion always resulted in a reduction of HA. This suggests that there may be an interaction between the proteins and HA in synovial fluid.

  21. Future Work • Test a new protease, either Protease K or Pronase E to digest the protein. • Perform rheological analysis on synovial before and after protein digestion to analyze the effects of the proteins on lubrication and shock absorption. • This would allow us to further determine the interaction between HA and the proteins in synovial fluid.

  22. Acknowledgments • Howard Hughes Medical Institute (HHMI) • URISC • Oregon State University • Dr. Skip Rochefort • Dr. Kevin Ahern • Dr. Jill Parker, OSU School of Veterinary Medicine • Shannon Cahill-Weisser, Project Assistant

  23. Viscosity • Indication of lubrication capabilities. Sheer Stress (Torque Measurement) Sheer Rate (Rotation Speed) Viscosity = Shear Stress Shear Rate

  24. Elasticity • A measure of shock absorption capabilities • Oscillating cone measures stored energy when fluid compressed.

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