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 Joint Cavity Articular Cartilage Articulating Bone Synovial Membrane 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.  Plasma proteins: albumin and globulin  Molecular weight range of 40 – 60 thousand g/mol. Proteins 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.waters.com/waters/partDetail.htm ?locale=en_US&partNumber=WAT045915 http://www.ap-lab.com/images/LS_setup.gif

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

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

14. Protein Digestion  Protease Bacillus 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 Digested Synovial Fluid Protein Molecular Weight: 6-8 x 10 4 g/mol Protein Molecular Weight: 3-6 x 10 3 g/mol -- Light Scatter -- Refractive Index -- Light Scatter -- Refractive Index

16. Protease Concentration  Increase to 4 units/mL Synovial Fluid  Same elution time = no gain in digestion  Low HA concentration Protein Molecular Weight: 3-6 x 10 3 g/mol -- Light Scatter -- Refractive Index

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. Protein Molecular Weight: 3-6 x 10 3 g/mol -- Light Scatter -- Refractive Index

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. Protein Molecular Weight: 3-6 x 10 3 g/mol -- Light Scatter -- Refractive Index

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. Protein Molecular Weight: 5-7 x 10 4 g/mol -- Light Scatter -- Refractive Index

20. Conclusion  The protease Bacillus 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.  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. Future Work

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

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

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