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.
Synovial Joint
Cavity
 Responsible for nutrient distribution, lubrication, and shock absorption.
Articular
Cartilage
Articulating
Bone
 Used for diagnosis of joint diseases.
Synovial
Membrane http:// edugen.wiley.com/edugen/student/mainfr.uni

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.

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

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

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

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

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

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

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

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

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

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.

Undigested Synovial Fluid
-Light Scatter
-Refractive Index
Protein Molecular Weight:
6-8 x 10 4 g/mol
Digested Synovial Fluid
-Light Scatter
-Refractive Index
Protein Molecular Weight:
3-6 x 10 3 g/mol

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

Removal of Dilution Step
 2 units protease/mL Synovial Fluid
-Light Scatter
-Refractive Index
Protein Molecular Weight:
3-6 x 10 3 g/mol
 Same elution time = no gain in digestion
 Low HA concentration continues. Possibly removed in filtration step.

Removal of Filtration Step
 2 units protease/mL Synovial Fluid
-Light Scatter
-Refractive Index
Protein Molecular Weight:
3-6 x 10 3 g/mol
 Same elution time = no gain in digestion
 Low HA concentration continues. Possibly removed in phenol-chloroform extraction.

Addition of Protease Only
 2 units protease/mL Synovial Fluid
-Light Scatter
-Refractive Index
Protein Molecular Weight:
5-7 x 10 4 g/mol
 Earlier elution time = Less digestion
 There is no change between the digested and undigested samples when treated with the protease.

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.

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.

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

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

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