Heparinic acid protein complex

Fischer and Schmitz were also interested in the interaction of heparin with proteins, such as casein and serum albumin. They reported that heparin appeared to shift the isoelectric point of the proteins to the acid side, probably by the formation of a molecular complex, and that combination with the protein occurred only near the isoelectric point and on the acid side." The complex was reversibly dissociated by the addition of alkali. These results are of irnportance in selecting conditions for the purification of crude heparin preparations, and constitute the basis for the extraction procedure developed by Charles and Scott, which has subsequently been used extensively. 

For the separation of heparin from heparin-protein complexes, Homan and Lens have developed a method which avoids the use of acid media. The heparin-protein complex is dissolved in aqueous solution at pH 7.5 and is extracted with phenol (which removes most of the protein). The method also facilitates the removal of colored impurities wLich are normally difficult to eliminate. Extraction of the heparin-protein-octylamine complex with phenol has been studied. "... 

Heparins and heparinoids are grouped in Table 3.2. Some are naturally occurring compounds, whilst others are derivatives prepared from heparins or from mucopolysaccharides. Semi-synthetic compounds have been prepared by degradation of natural polysaccharides followed by sulphation with chlorsulphonic acid or methyl sulphate . The common activities shown by the heparins and heparinoids—complexing with organic bases and proteins, antilipaemic activity and anticoagulant activity—are also shown by various sulphonic acid dyes and by polyphosphates. The table is completed with a list of those preparations that have been issued to provide depot preparations of heparins and heparinoids. 

In view of the uncertainty as to the existence of a heparin-protein complex, Idndahl et al. (1965) analyzed a number of heparin samples for the presence of amino acids. It was found that heparin which had been prepared by use of mild methods, essentially involving only treatment with proteolytic enzymes, contained residual amino acids with serine as the main component. In some preparations serine was the only amino acid found in significant amounts. Samples prepared under more drastic conditions, including alkali extraction or bleaching, contained only traces of amino acids. These findings thus suggest that heparin does occur in the tissues as a covalently bound complex with protein. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

Although the order of affinity of PF-4 for different glycosaminogly-cans, and dissociation of their complexes with salts, are typical of nonspecific, electrostatic interactions, PF-4 is not strictly a cationic protein.452 It is probable that heparin binds to clusters of basic amino acids (two lysine pairs) near the carboxyl terminal of a polypeptide chain that has an overall preponderance of acidic amino acid residues.457 High-molecular-weight heparin species can bind two PF-4 molecules, with formation of complexes 10 to 100 times as strong as those with antithrombin.217... 

Synthesis Histamine is an amine formed by the decarboxylation of the amino acid histidine (Figure 40.3). This process occurs primarily in the mast cells, basophils, and in the lungs, skin, and gastrointestinal mucosa—the same tissues in which histamine is stored. In mast cells, histamine is stored in granules as an inactive complex composed of histamine and the polysulfated anion, heparin, along with an anionic protein. If histamine is not stored, it is rapidly inactivated by amine oxidase enzymes. 

The term heparin has not been used consistently since its introduction it usually implies the sodium salt of the sulfated polysaccharide, but many other salts are known. Perhaps a more logical name would be hepariiiic acid the sodium salt would then be termed sodium heparinate, and the naturally occurring complex which contains the anticoagulant, protein heparinate. In this Chapter, the term heparin signifies the sodium salt, unless otherwise qualified. 

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