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Determination of the Role of Proteins
in the Physical Properties of
Equine Synovial Fluid
By: Shannon Cahill-Weisser
Advisor: Dr. Skip Rochefort
Oregon State University School of Chemical, Biological,
and Environmental Engineering
HHMI Summer 2010
reboundsportsphysio.com.au/information/glucosamine
The Problem

21.6% of U.S. adults reported doctor
diagnosed arthritis (2003-2005)
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8.8% of U.S. adults reported activity
limitations (2003-2005)
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Arthritis cost the United States
$128 Billion in 2003
Synovial Fluid
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Viscoelastic fluid in
diarthrotic joints
Keeps join lubricated,
absorbs shock
Supplies nutrients to
cartilage
Largest molecules
present are proteins
and hyaluronic acid
http://www.nytimes.com/imagepages/2007/08/01/
health/adam/19309Kneejoint.html
Equine Joint Atlas
http://oregonstate.edu/dept/biochem/hhmi/undergradresearch/2005/index.html
Hyaluronic Acid (HA)
Key anionic glycosaminoglycan (GAG)
polymer in synovial fluid
 Molecular weight 0.2-10 million Daltons
 Conc. range in synovial fluid 2-4 mg/mL
 Found in lower concentrations with lower
molecular weight in diseased joints

www.valleyvet.com/ct_detail.html?pgguid
=b3a23e6a-62a1-4e9b-9700-d54971e2da5f
http://oregonstate.edu/dept/biochem/
hhmiundergradresearch/2009/program.htm
Proteins
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Plasma proteins such as albumin and globulin
Serum albumin ~ 66 thousand Daltons, most
prominent protein in synovial fluid
Globulins have a wider molecular weight range
Total protein conc. in synovial fluid 18-48 mg/mL
Studies show they may have electrostatic
interactions with HA, forming protein-GAG
complexes
May also form aggregated protein structures
Research Plan

Objectives: Characterize the interactions
between synovial fluid proteins and hyaluronic
acid.

Hypothesis: Interaction exists that affects the
rheological properties of synovial fluid.

Plan: Refine method of digestion/extraction of
synovial fluid proteins without removal or
compromise of HA.
Size Exclusion ChromatographyMulti Angle Laser Light Scattering
http://www.ap-lab.com/images/LS_setup.gif
Size Exclusion Columns
Light Scattering Geometry
media.wiley.com/CurrentProtocols/PS/ps0708/ps0708-fig-0001-1-full.gif
http://www.viscotek.com/images/Theory/separate.jpg
Laboratory SEC-MALLS
Polymer Labs Aquagel-OH SEC Columns
Wyatt Dawn EOS
Hitachi HPLC Pump
Hitachi DRI
Digestion and Extraction

 Lower
HA peak in digested sample, implying loss of HA
 Run to run inconsistencies in protein Mw
Undigested
Digested
Peak ID - 5 mo
Peak ID - 5 mo
0.4
0.4
LS #11
AUX2
0.3
0.2
0.1
12
0.0
-0.1
20
LS #11
AUX2
0.3
LS #11, AUX2

Breaks proteins into amino acids
Pieces removed through phenol-chloroform
extraction
Results:
LS #11, AUX2

40
60
80
T ime (min)
100
120
140
0.2
0.1
0.0
-0.1
20
40
60
80
T ime (min)
100
120
140
Samples of Study
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Sample 1: RLTR,
2005,Normal
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HA Elution: 33.994 min
HA Mw: 2.65E+06 Da
Protein Elution: 47.328 min
Protein Mw: 7.61E+04 Da
Sample 2: Left Stifle, 2005,
Severe Trauma Fracture
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Peak 1 Elution: 43.921 min
Peak 1 Mw: 7.288E+05 Da
Peak 2 Elution: 47.513 min
Peak 2 Mw: 8.490E+04 Da
Elution Time Comparisons
Gel Electrophoresis Procedure

Bradford Assay:
optical density
measurement of
coomassie blue
bound proteins
Run on SDSpolyacylamide gels
Protein Concentrations
20
18
16
14
12
10
8
6
4
2
0
9MQH Normal
Conc. (mg/mL)
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Severe Trauma
Fracture
Literature Protein
Conc. 1
Literature Protein
Conc. 2
1
Samples
Gel Electrophoresis
5 mo. old Colt, Normal
Severe Trauma Fracture
9 mo. Normal
Future…
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Run gel configured for glycosaminoglycan
analysis
 Fluorophore-Assisted
Carbohydrate
Electrophoresis
 Try toluidine blue and other dyes

Examine synovial fluid for degraded HA
pieces before and after digestion.
 Can
show HA sizes at lower end of range
References
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Calabro, Anthony, Benavides, Maria, Tammi, Markku, Hascall, Vincent C., and Midura, Ronald
J. (1999). “Microanalysis of Enzyme Digests of Hyaluronan and Chondroitin/Dermatan
Sulfate by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)”. Glycobiology 2000,
vol. 10 no. 3, pp.273-281.
Centers for Disease Control and Prevention (2006 and 2007). “Prevalence of DoctorDiagnosed Arthritis and Arthritis-Attributable Activity Limitation, 2003—2005”. Morbidity and
Mortality Weekly Report (MMWR), 2006: 55(40); 1089-1092 and 2007/ 56(03); 55. Available
at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5540a2.htm. Accessed: August 6, 2009.
Bhusari, Amol. (2007). Computational and Experimental Investigation of the Role of
Hyaluronic Acid-Protein Interactions in the Rheology of Synovial Fluid. Texas Tech University.
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.
Hans, Tricia, Latimer, Kenneth S., LeRoy, Bruce E., Bain, Perry J., Tarpley, Heather L., Frank,
Paul M. (2004). “Synovial Fluid Findings in Degenerative Joint Disease”.
http://www.vet.uga.edu/vpp/clerk/hans/index.php. Accessed: September, 2010
Leiske, Danielle Lurisa. (2004). Molecular and Rheological Characterization of Sodium
Hyaluronate (HA) and Equine Synovial Fluid. Oregon State University.
Sigma Aldrich. (2010). “Albumin from Bovine Serum”.
http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=A2153|SIGMA&N5=SEA
RCH_CONCAT_PNO|BRAND_KEY&F=SPEC. Accessed: September, 2010.
Yelin, E (PhD), Cisternas, M (MA), Foreman, A (MA), Pasta, D (MS), Murphy L (PhD),
Helmick, CG (MD) (2007). “National and State Medical Expenditures and Lost Earnings
Attributable to Arthritis and other Rheumatic Conditions—United States, 2003”. MMWR 2007:
56(01); 4-7. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5601a2.htm.
Accessed: August, 2009.
Acknowledgements
Howard Hughes Medical Institute
 Pete and Rosalie Johnson Internship
 Dr. Skip Rochefort
 Dr. Jill Parker, OSU Veterinary School
 Dr. Weis, Angela Poole, and Emilie Dicks
 Talia Helman, Dan Foster, Karl Schilke,
Marsha Lampi, Marshall Lake, Danielle
Leiske
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