Between my second and third year of Mechanical Engineering at the Schulich School of Engineering, I was fortunate enough to receive an NSERC Undergraduate Research Award (USRA) to work with Dr. Tannin Schmidt and a fellow student Cecilia Alvarez on a project involving the development of biotheraputics for osteoarthritis treatment.
We were interested in the lubricating properties of a protein found in the synovial fluid (SF) in your joints, Proteoglycan-4 (PRG4, or Lubricin), as it contributes to the overall maintenance and integrity of the joint. At the time, techniques used to purify this protein from the native SF relied on the use of highly concentrated salt solutions to aid in separation of the various components.
Salt tends to denature proteins – causes structural changes that can alter their mechanical properties. You can test this in your kitchen by cracking an egg into a pan and immediately salting it versus waiting for it to cook before salting. The former case will result in a runnier egg. It had been shown that PRG4 existed in two forms, a Monomer and a Multimer (links of monomers), and that treatment of the PRG4 protein with heavy salt solution diminished it’s lubricating function, but a link between the structure and function of the protein had not been established.
Using biochemical methods, we separated the PRG4 protein from bovine synovial fluid and used it as the lubricant in biomechanical friction tests performed with a Bose Electroforce machine. I would walk to a nearby butcher shop who were used to getting bizarre research requests, and spend a day in the lab with a scalpel, bone saw and drill press creating annulus and core samples from bovine stifle joints.
We could place these bone/cartilage plugs into the Bose Electroforce and lubricate them with different preparations of PRG4 proteins, compared to the synovial fluid control.
Our research, presented by myself at the 2010 Orthopaedic Research Society’s international conference, showed that the heavy salts used in separating the PRG4 molecule from synovial fluid were altering the structure AND the function of the PRG4 protein, meaning that researchers aiming to study the protein should find other methods of purifying it.