The assay is based on the following reaction:
Hyaluronic acid ------> Acetylglucosamine
An anhydro sugar is first formed from N-acetylglucosamine, followed by the conversion of the sugar into its furan derivative, as a result of increasing the acidity of the solution. This furan derivative then reacts with p-dimethylamino-benzaldehyde to form a colored complex, which is measured spectrophotometrically at 585 nm. Detailed procedures for the assay of hyaluronidase are available in Methods of Enzymatic Analysis, Bergmeyer, H.U., ed., Vol. 2, pp. 944-948, Academic Press, New York, 1974.