Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions

Abstract

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage was studied by equilibrium dialysis in dilute solution. Each proteoglycan monomer binds only 1 hyaluronate oligosaccharide molecule [containing .apprx. 18 saccharide residues .**GRAPHIC**. and of number-average molecule weight (.hivin.Mn) 3750]. Kd for interaction between proteoglycan monomer and oligosaccharide .**GRAPHIC**. is 3 .times. 10-8 M at 6.degree. C at I [ionic strength] 0.15-0.5, pH 7.4. Kd has little dependence on temperature, so that Kd at 54.degree. C is 3 .times. 10-7 M under the same conditions. The aggregatability is high at 6.degree. C, falls significantly at 54.degree. C, but much of it can be recovered on cooling to 6.degree. C again, demonstrating reversible denaturation. A method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods. A hyaluronate oligosaccharide, .**GRAPHIC**. (.hivin.Mn 11,000), could bind more than 1 proteoglycan molecule. Ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (MW 670,000), an average Kd of 2 .times. 107 M fits the data in 0.5 M guanidine hydrochloride at 20.degree. C.