A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyahrronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supematant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with ['Hlhyaluronate, followed by an equal volume of saturated (NH&SO,, which precipitated the hyaluronectin and any [3H]hyaluronate associated with it, but left free ['Hlhyaluronate in solution.
The mixture was then centrifuged, and the amount of bound ['Hlhyaluronate in the precipitate was determined. Using this assay, we found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity (& = 5.7 X lO-'O M), and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, we used a competition assay in which the binding ofa set amount of [3H]hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. We have found that this method is sensitive to 1 ILcg or less of hyaluronate, and is unaffected by the presence of prOtt%S.