title: Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.
abstract: The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.
authors.affiliation: Polytechnic Institute of New York University, Brooklyn, NY 11201, USA. mcowman@poly.edu
authors.forename: Mary K
authors.initials: MK
authors.lastname: Cowman
authors.forename: Cherry C
authors.initials: CC
authors.lastname: Chen
authors.forename: Monika
authors.initials: M
authors.lastname: Pandya
authors.forename: Han
authors.initials: H
authors.lastname: Yuan
authors.forename: Dianne
authors.initials: D
authors.lastname: Ramkishun
authors.forename: Jaclyn
authors.initials: J
authors.lastname: LoBello
authors.forename: Shardul
authors.initials: S
authors.lastname: Bhilocha
authors.forename: Sparkle
authors.initials: S
authors.lastname: Russell-Puleri
authors.forename: Eraldi
authors.initials: E
authors.lastname: Skendaj
authors.forename: Jovan
authors.initials: J
authors.lastname: Mijovic
authors.forename: Wei
authors.initials: W
authors.lastname: Jing
mesh_terms: D002138:Calibration; D003720:Densitometry; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006820:Hyaluronic Acid / Q000032:analysis* / Q000737:chemistry*; D008970:Molecular Weight; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D014783:Viscosity
chemical_list: D006820:Hyaluronic Acid