Hyaluronic acid complex with protein

In most connective tissues of animals, the acidic mucopolysaccharides are complexed with protein or peptide residues. Little is known about the structure of these complexes, and the term mucopolysaccharide is therefore best applied only to the pure polysaccharide. When the latter is complexed with protein, it has been suggested (J5) that a noncommittal term such as hyaluronic acid-protein complex should be used. Many of the names originally assigned to the acidic mucopolysaccharides have since been revised (J5) in an effort to systematize the nomenclature. The more systematic names proposed by Jeanloz (J5) will generally be used throughout this review, but whenever possible synonymic names have been given. 

A method for hyaluronic acid extraction by aqueous-alcohol mixture, containing 5-25% n-propyl, iso-propyl or tert-butyl alcohol, 2M calcium chloride (of cartilage origin) is described. In order to isolate hyaluronan from its complexes with proteins, a homogenized tissue is treated with proteolytic enzymes (pepsin, trypsin, papain and/or pronase) and aqueous-organic mixtures. But in most methods these procedures are included in tlie treatment of the primary extracts. 

L-arabinose, have also been reported to be in direct linkage with a protein. L-Arabinose may also be linked to L-serine and L-threonine in a hyaluronic acid-polypeptide complex from ox vitreous-humor.750 D-Mannose is reported to be glycosidically linked in a glucoamylase [(l->4)-a-D-glucan glucohydrolase, EC 3.2.1.3] from Aspergillus... 

The classical thermodynamic and kinetic model is that of a rigid sphere impenetrable by water. A spherical geometry has been observed in many polysaccharide systems, notably hyaluronic acid-protein complexes (Ogston and Stainer, 1951), dispersed gum arabic (Whistler, 1993), and spray-dried ungelatinized starch granules (Zhao and Whistler, 1994). Spherulites of short-chain amylose were obtained by precipitation with 30% water-ethanol (Ring et al., 1987), and spherulites of synthetic polymers were obtained... 

Since protein complex formation and Ca2+ are critical to cell fixation within a tissue, dissociation media usually contain some type of proteolytic enzyme and the Ca2+ chelator, EDTA. The proteolytic enzyme can be of general specificity, such as trypsin, or can be a more targeted enzyme, such as a collagenase selective for the collagen-type characteristic of the tissue of interest. Hyaluronidase has been also used with matrix rich in hyaluronic acid, such as for isolation of duodenal entero-cytes. In all cases, the appropriate incubation times and concentrations to achieve cell dispersal, but retain high viability, need to be determined empirically. One factor... 

The above results suggest intimate association of proteins and proteoglycans in desmosomes and in surface coats of cells. A possible example of this kind of association is provided by studies of films made with various proportions of protein and proteoglycan. Mixtures of 2 parts gelatin and 1 part hyaluronic acid are much more resistant to diflFusion of various substances than other combinations of these ingredients (40). Perhaps intercellular discs represent mechanically dense and viscid complexes of similar nature. The disappearance of desmosomal components into reconstituted intercellular cement suggests that they have similar chemical components but different stoichiometry. 

Other possible physiological functions of hyaluronic acid may be associated with its macroanionic nature. Aldrich (A4), for example, observed that Ca++ was strongly associated with a hyaluronic acid-protein complex, and was not readily displaced by high concentrations of univalent cations. A possible role for hyaluronic acid in regulating the concentrations of Na+ and K+ in nerve fibers has also been suggested (Al). 

The current method for the hyaluronidase assay described in the United States Pharmacopeia (USP) [132] is based on the inability of hydrolyzed potassium hyaluronate to form a complex precipitate with proteins from added serum, reflected in a decreased turbidity of the reaction mixture (measured after 30 min). The method is, from the enzymological point of view, not well defined since it does not actually evaluate the kinetics of the hydrolysis of the substrate. An assay with end-point determination is only valid if the reaction rate does not change during this reaction time. We found that only with the two lowest test concentrations (0.15 and 0.3 IU) was this condition fulfilled, while with the three higher test concentrations the reaction is not linear. Commercially available hyaluronates can be contaminated with chondroitin sulfates. They are more acidic than hyaluronic acid itself and hence can form better protein complexes and influence the turbidity. In a suitability test of the USP [133], the substrate must pass both an inhibitor content test and a turbidity-production test. The assumption is made that... 

The other major component of an articular joint is synovial fluid, named by Paracelsus after synovia (egg-white). It is essentially a dialysate of blood plasma with added hyaluronic acid. Synovial fluid contains complex proteins, polysaccharides, and other compounds. Its chief constituent is water (approximately 85%). Synovial fluid functions as a joint lubricant, nutrient for cartilage, and carrier for waste products. 

It has already been shown and discussed — at least in in vitro tests with articular cartilage — that compounds which reduce friction do not necessarily reduce wear the latter was suggested as being more important [10]. It maybe helpful first of all to emphasize once again that friction and wear are different phenomena. Furthermore, certain constituents of synovial fluid (e.g., Swann s Lubricating Glycoprotein) may act to reduce friction in synovial joints while other constituents (e.g., Swann s protein complex or hyaluronic acid) may act to reduce cartilage wear. 

The relatively small number of culture supernatant proteins (CSPs), compared with proteins in the cytoplasm, simplifies proteomic analysis. However, several aspects can make two-dimensional gel electrophoresis (2-DE) of CSPs challenging (6). These include (1) the presence of basic CSPs, which can become insoluble during isoelectric focusing, (2) the presence of hyaluronic acid, which can interfere with isoelectric focusing, (3) the presence of peptides in complex media, such as Todd-Hewitt broth, and (4) the abundance of specific exoproteins such as the cysteine protease SpeB, which can mask less abundant proteins (7). 

---