EXTRACTION AND BINDING CHARACTERIZATION OF HYALURONIC ACID BINDING PROTEINS FROM BOVINE NASAL CARTILAGE

Open Access
Author:
Weaver, Bret Michael
Graduate Program:
Physiology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
None
Committee Members:
  • Herbert Herling Lipowsky, Thesis Advisor
Keywords:
  • Bovine Nasal Cartilage
  • Hyaluronic Acid Binding Proteins
Abstract:
The current project was undertaken to optimize the extraction of hyaluronic-acid binding proteins (HABPs) from bovine nasal cartilage (BNC). An incubation pull-down assay was devised by mixing various concentrations of trypsinized BNC with a constant amount of functionalized sepharose beads conjugated with hyaluronic acid (HA). Following incubation of BNC digests with the HA-derived beads, a fractional binding profile was established for a range of digest-to-bead proportions. Elution of the beads with 2M NaCl and 4M GCl showed most HABP binding activity coming off with 2M NaCl. Total protein and fractional binding values were determined with Bradford assay and SDS-PAGE electrophoresis, respectively. From these results, a mass elution scheme was prepared to follow a step-wise washing pattern proportional to ionic strength. This allowed protein yield and specific binding amounts to be calculated. Conjugation efficiency of the derived sepharose beads was determined by measuring the difference in refractive index (RI) between the supernatant and the initial solution concentration of added 64kDa HA. The refractive indices of a stock solution of HA were used to construct a standard curve to calculate HA (w/v) % of the supernatant. Calculation of HABP binding values was determined using the protein concentrations eluted with 2M NaCl as they were found to be statistically more significant than the 4M GCl rinses (P<0.01). A minimum of three percent conjugation was calculated from a standard conjugation assay with non-degraded 64kDa HA and functionalized sepharose beads. Trypsinized HABPs from BNC were found to have a MWw of 53.4 kDa, a Kd of 7.235x10-8M and a total binding-site concentration ([TBS]) of 1.775x10-6M with most of the binding activity residing in the 47kDa link protein. Total specific binding activity gave a yield of 1.18% deduced from the mass-elution procedure. iii The current project was focused around methods and results previously reported (Tengblad, 1978). Using conventional liquid affinity chromatography, specific binding activity was washed out in the 4M GCl fraction. Two species are commonly found to bind to HA after being extracted from trypsinized BNC: a 45kDa link protein and a 90kDa proteoglycan monomer. Three bands were analyzed with the current incubation pull-down method that contributed to the overall calculation of the weighted molecular-weight average: 97kDa, 65kDa and 47kDa. This approach was derived from previous reports, and MW differences were due to the higher concentration of acrylamide used to run the reducing PAGE. Liquid chromatography remains to be the best procedure in order to enhance the purity of the sample for proper in-vivo experimentation and interpretation of conjugative biochemical results.