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

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) 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; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.