Plasma proteins, ethanol fractionation

Immune Globulin (IG) IG is a sterile solution containing antibodies from human blood. It is obtained by cold ethanol fractionation of large pools of blood plasma and contains 15-18 percent protein. Intended for intramuscular administration, IG is primarily indicated for routine maintenance of immunity of certain immunodeficient persons and for passive immunity against measles and hepatitis. IG does not transmit hepatitis B virus, human immunodeficiency virus (HIV), or other infectious diseases. 

The iron-binding protein of serum transferrin was found in fraction IV-3,4 of human plasma when the plasma was fractionated by low temperature ethanol fractionation procedures (31, 116). By further subfractionations, serum transferrin could be concentrated in Cohn fraction IV-7 (30, 125, 126). Cohn (30) first reported the properties of the isolated protein, which he called the 3i metal-binding protein since the protein had been found to bind copper, and possibly zinc, as well as iron. Holm-berg and Laurell (66) proposed that the protein be called transferrin on the basis that the principal function of the protein was associated with the transport of iron in serum and that it was not the major copperbinding protein in human serum. 

An important property of the protein metal complexes is that their solubilities are particularly influenced by the dielectric constant of the medium. Thus, a moderate reduction of the dielectric constant of an aqueous solution will precipitate the Ba or Zn salts of many proteins which are readily soluble in the aqueous solutions. This property has been used for the fractionation of the plasma proteins. The use of barium and zinc salts not only gave superior fractionation, but also allowed the use of lower ethanol concentrations with diminished risk of denaturation. 

In the late 1940s, a method was developed (Cohn et al. 1946) for the fractionation of proteins from blood plasma by means of ethanol (an anti-solvent) addition to aqueous solutions at specified conditions of temperature, pH, and ionic strength. Briefly, a series of five successive batch ethanol precipitations were used to prepare fractions of fibrinogens, globulins, and albumin. The conditions used in each step are in Table 11.1 along with the major protein(s) precipitated at each step. More recently, a method employing a series of MSMPR precipitators has been reported (Chang 1988). 

TABLE 11.1 Conditions for Ethanol Fractionation of Plasma Proteins... 

To be clinically useful, dextrans have to be partially hydrolyzed to a molecular weight range comparable to that of plasma proteins. Furthermore, ethanol fractionation and extensive purification is required. The clinical dextrans used today are prepared from native dextran produced by the Leuconostoc mesenteroides NRRL B 512 strain (see Fig. 2). This dextran is only slightly branched, possessing 1 side chain for every 20 glucose units. Of the side chains, 85% comprise only 1-2 glucose units (Larm et al. 1971). 

Multi-variable or cold-ethanol precipitation techniques yielded plasma protein fractions rich in lipoprotein (Oncley et al. 1949). These isolation procedures were extremely important. They demonstrated that physical properties such as solubility could be studied with lipoproteins as well as other proteins. Electrophoretic mobilities and specific refractive increments of lipoproteins were shown to be measurable properties (Armstrong et al. 1947 a and 1947 b). A detailed analysis of lipoprotein composition was undertaken (Oncley et al. 1950). 

Factor VIII, immunoglobulin, and albumin are all held as protein precipitates, the first as cryoprecipitate and the others as the Cohn fractions FI + II + III (or FII + III) and FIV + V (or FV), respectively (Table 7, Fig. 2). Similarly, Fractions FIVj + FIV can provide an intermediate product for the preparation of antithrombin III and a-1-proteinase inhibitor. This abiUty to reduce plasma to a number of compact, stable, intermediate products, together with the bacteriacidal properties of cold-ethanol, are the principal reasons these methods are stiU used industrially. 

Tincture of the dried seed, on agar plate at a concentration of 30 p,L/disc, was inactive on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Extract of 10 g plant material in 100 mL ethanol was used b Anticoagulation activity. Serpin BSZx (an inhibitor of trypsin and chemotrypsin) inhibited thrombin, plasma kallikrein, factor Vlla/tissue factor, and factor Xa at heparin-independent association rates. Only factor Xa turned a significant fraction of BSZx over as substrate. Activated protein C and leukocyte elastase were slowly inhibited by BSZx, whereas factor Xlla, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein, and elastase were not or only weakly affected. Trypsin from Fusarium was not inhibited, while interaction with subtilisin Carlsberg and Novo was rapid, but most BSZx was cleaved as a substrate L... 

The immunoglobulins are obtained from the plasma pools by fractionation methods that are based on ethanol precipitation in the cold with rigorous control of protein concentration, pH and ionic strength (Cohn et al., 1946). The variation of... 

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