Hyaluronic acid enzymic analysis

A report by Liu and coworkers described layer-by-layer (LBL) coatings that have been assembled on the inner surfaces of the microchip [90]. Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. Trypsin was adsorbed in the multilayer membrane composed of CS/HA assembled multilayers. The resulting peptide analysis has been carried out by MALDI-TOF-MS. The maximum proteolytic velocity of the adsorbed trypsin was 600 mM/min/[ig, thousands of times faster than that in solution. BSA, MYO, and Cyt-c were used as model substrates for the tryptic digestion. The standard proteins were identifled at a low femtomole per analysis at a concentration of 0.5 ng/pl with the digestion time <5 s. This simple technique may offer a potential solution for low-level protein analysis. 

One of the earliest methods of detecting hyaluronic acid was the formation of a so-called mucin clot in presence of proteins, by addition of acetic acid to fluids or tissue extracts. Detection by mecisurements of the decrease of viscosity, after addition of hyaluronidase, is not reliable, because the pure enz)nne is not available, and the mixture of enzymes used also degrades the chondroitin sulfates. Definite identification by chemical means requires separation on a microscale, using selective adsorption on a column, or fractionation by electrophoresis, determination of the ph3 cal characteristics, and analysis of the components. 

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