Determining the Role of Proteins in the Molecular Properties of

Equine Synovial Fluid

Marsha Lampi

Advisor: Dr. Skip RochefortOregon State University

School of Chemical, Biological and Environmental EngineeringSummer 2009

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

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.

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

Equine Synovial Fluid

Stifle (knee)

Hock (ankle)

http://www.ucmp.berkeley.edu/education/lessons/xenosmilus/skeletal_res_manual2.html

Objective

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

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.

Analysis of Molecular

Composition

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

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, Io

Polymer SolutionLight Source

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

HA Peak

Protein Peak

Before digestion, both the HA and protein peaks are detected.

GPC-MALLS Graph

-- Light Scatter-- Refractive Index

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

Digested Synovial Fluid

Protein Molecular Weight:6-8 x 104 g/mol

Protein Molecular Weight:3-6 x 103 g/mol

-- Light Scatter-- Refractive Index

-- Light Scatter-- Refractive Index

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 103 g/mol

-- Light Scatter-- Refractive Index

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 103 g/mol

-- Light Scatter-- Refractive Index

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 103 g/mol

-- Light Scatter-- Refractive Index

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 104 g/mol

-- Light Scatter-- Refractive Index

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.

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

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.

Viscosity = Shear Stress Shear Rate

Sheer Rate(Rotation Speed)

Sheer Stress(Torque Measurement)

Elasticity

A measure of shock absorption capabilities

Oscillating cone measures stored energy when fluid compressed.