An easy and rapid method for the purification and characterization of hyaluronan from synovial fluid has been developed. Lipids were removed by filtration through a hydrophobic filter prior to the removal of proteins by phenol-chloroform extraction. The hyaluronan recovery was 95%, as measured by three different methods. The average molecular weight of hyaluronan did not change during the purification. Furthermore, it was found that an optimized enzymatic protein digestion of pathological human synovial fluid prior to filtration yielded up to five times more hyaluronan recovered. In addition, the molecular weight of hyaluronan from synovial fluid not digested with pronase E was apparently higher because of the presence of aggregates. After the purification of hyaluronan (ca. 15 min), a single size-exclusion chromatography step allowed the simultaneous determination of its concentration and the reasonable estimate of its average molecular weight and molecular weight distribution curve. The logarithm of the molecular weight showed a linear dependence on the size-exclusion chromatography elution volume for hyaluronan in the molecular weight range 2.0 x 10(6)-1.0 x 10(4). The removal of proteins allowed the determination of fairly low-molecular-weight fractions of hyaluronan, compared to untreated samples for which hyaluronan and protein peaks partially overlap.
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