Protein from blood plasma

SAMPLE EXTRACTION. For removal of proteins from blood plasma with picrate, 5 cm3 of blood plasma is placed in a 125-cin Erlen-meyer flask with 25 cm3 of 10% aqueous picric acid and stirred... 

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). 

Surface layer-MALDI-MS has been developed specifically to identify proteins adsorbed onto biomaterial surfaces. Although the experimental approach for this technique is analogous to traditional MALDI-ToF MS, surface layer-MALDI-MS does not require protein isolation from the biomaterial surface, because the protein-adsorbed surface is submerged directly in a matrix solvent for crystallization (Griesser et al., 2004). For example, surface-MALDI-MS can be used to identify which proteins from blood plasma adsorb onto a biomaterial surface directly off the original material surface, such as polyurethane, as shown in Fig. 5.14 (Oleschuk et al., 2000). The acquired spectra show clear peaks, indicating different proteins with a distinct mass. Each of these proteins can be identified by comparing the experimentally determined masses with those in the literature. 

Carotenoids are also present in animals, including humans, where they are selectively absorbed from diet (Furr and Clark 1997). Because of their hydrophobic nature, carotenoids are located either in the lipid bilayer portion of membranes or form complexes with specific proteins, usually associated with membranes. In animals and humans, dietary carotenoids are transported in blood plasma as complexes with lipoproteins (Krinsky et al. 1958, Tso 1981) and accumulate in various organs and tissues (Parker 1989, Kaplan et al. 1990, Tanumihardjo et al. 1990, Schmitz et al. 1991, Khachik et al. 1998, Hata et al. 2000). The highest concentration of carotenoids can be found in the eye retina of primates. In the retina of the human eye, where two dipolar carotenoids, lutein and zeaxan-thin, selectively accumulate from blood plasma, this concentration can reach as high as 0.1-1.0mM (Snodderly et al. 1984, Landrum et al. 1999). It has been shown that in the retina, carotenoids are associated with lipid bilayer membranes (Sommerburg et al. 1999, Rapp et al. 2000) although, some macular carotenoids may be connected to specific membrane-bound proteins (Bernstein et al. 1997, Bhosale et al. 2004). 

This receptor-mediated endocytotic pathway has been especially well studied in the uptake of iron from blood plasma. Iron, because of its very low-solubility product (< 1(T17 at pH 7.4), is transported in plasma bound to the iron-binding protein transferrin. Two Fe3+ ions bind to each transferrin molecule. Entry into... 

Some dmgs are bound to plasma proteins in blood. Plasma protein levels in blood may be decreased in the elderly, but this is most often not clinically relevant since a drug s elimination increases when the free, unbound drug concentration is enhanced (Turnheim 1998). The plasma albumin level may however be markedly decreased in elderly suffering from malnutrition or severe disease. For those patients the concentration of the free unbound drug can reach toxic levels (Waiter-Sack and Klotz 1996). 

Sarnatskaya V, Yushko L, Nikolaev A et al (2007) New approaches to the removal of protein-bound toxins from blood plasma of uremic patients. Artif Cells Blood Substit ImmobU Biotechnol 3 287-308... 

Serum albumin is a natural transporter of hydrophobic metabolites it binds toxic ligands reversibly, and if the ligand can be removed from the complex then the melting curve of unloaded albumin should return to that of pure protein. However this remains a difficult task, and neither exhaustive dialysis, nor the use of conventional carbonic sorbents, influence the shape of melting curves of albumin isolated from blood plasma of uremic patients and patients with hepatic insufficiency (Fig. 29.3) [9]. 

Table 4 Percentage Occurrence of the Major Complexes (Non-protein) in Blood Plasma from Computer Simulation -1 ...

Serum albumin is the most plentiful protein in blood plasma. Each molecule can carry seven fatty acid molecules. They bind in deep crevices in the protein, burying their carbon-rich chains away from the surrounding water. Serum albumin also binds to many other water-insoluble molecules. In particular, serum albumin binds to many drug molecules and can strongly affect the way they are delivered through the body. 

Data presented in previous sections revealed that the concentration of FFAs in plasma may reach 2.0 mM during exercise. How is this possible when the highest attainable concentration in water is only about 0,1 mM This problem was resolved by nature by use of albumin as a vehicle for the transport of FFAs within the circulation. Albumin constitutes about 60% of the protein of blood plasma. It is a major carrier of FFAs, other metabolites, hormones, and drugs- Serum albumin has the capadty to carry several fatty adds. Figure 4.45 shows results from an experiment usingpurificdalbumin.Thenumberoffattyacid molecules bound per protein molecule is plotted versus the concentration of unbound fatty acids in solution. The study, conducted with lauric acid (12 carbons) and myristic add (14 carbons), demonstrates that one protein molecule is able to bind at least 8 or 9 molecules of fatty acid. Albumin has a molecular weight of 69 kDa and occurs in human plasma at a concentration of about 0.6 mM (40 mg/ml) (Halliwell, 1988). 

The proteins most amenable to routine laboratory evaluation die those in blood, urine, CSF amniotic fluid, saliva, feces, and peritoneal or pleural fluids. With few exceptions, the proteins found in all of these are derived from blood plasma. The following discussion is limited to (1) the most abundant plasma proteins, (2) changes of their concentrations in the most accessible body fluids, and (3) a few of the analytical techniques used to measure them. 

In the meantime, in 1947, Laurell and Ingelman[17] had independently purified the red protein from pig plasma and in the same year proposed the name transferrin which has since been adopted as the generic name of the proteins of this family serotransferrin (instead of siderophilin) present in blood and some external secretions, ovotransferrin (instead of conalbumin) in avian egg-white, lactotransferrin (also called lactoferrin) from milk, external secretions and leukocytes and melanotransferrin (instead of p97) in melanocyte and normal cell plasma membrane. A dozen mammalian and some frog, fish and insect serotransferrins were later isolated and characterized. 

Many different types of proteins are glycoproteins. For example, stractural proteins such as collagen, proteins found in mucous secretions, immunoglobulins, folhcle-stimu-lating hormone and thyroid-stimulating hormone, interferon (an antiviral protein), and blood plasma proteins are all glycoproteins. One of the functions of the polysaccharide chain is to act as a receptor site on the cell surface in order to transmit signals from hormones and other molecules across the cell membrane into the cell. The carbohydrates on the surfaces of cells also serve as points of attachment for other cells, vimses, and toxins. 

The adsorption of plasma proteins to polymers precedes the interaction of blood cells with the surfaces, and therefore, is likely to be an important initial event in the response of blood to polymers (25, 29). At present, however, little is known about the adsorbed protein layer, even though it has been studied in some detail in recent years (30-36). Because protein adsorption from blood plasma is a competitive process, differences in the adsorbed layer on different polymer substrates could be a primary cause of differences in thrombogenicity. Previous studies of the composition of the adsorbed protein layer have employed 12oI-labeled protein added to plasma (37-39), antibody binding (34) to detect individual proteins, or electrophoretic analysis of detergent-elutable proteins (17, 33, 35). The procedure used in this study does not require the large surface areas used in previous work (35), nor does it rely on incorporation of radiolabels (36) into adsorbed protein. Instead, a staining method at least 100-fold more sensitive than these other techniques has been used. 

Arteriovenous (AV) difference studies and mammary blood flow measurements (Chapter 1) have shown that in both ruminants and non-ruminants, amino acids for milk protein synthesis are obtained ultimately from blood plasma but that some interconversions occur. The amino acids can be divided into two major groups ... 

We begin with some comments on the various experimental methods used in our studies. Investigations of single proteins in buffer are then discussed, including kinetics and isotherms, reversibility, denaturation, and the structural status of adsorbed proteins. Results of competitive adsorption studies using mixtures of proteins in buffer are then described. We next discuss our more recent studies of protein adsorption from blood plasma using both radiolabeled proteins and elution techniques. Finally, data on the effect of red blood cells on protein adsorption are summarized. 

Incubation of a protein fraction from blood plasma with trypsin gives rise to peptides with conspicuous biological effects. Pain, dilation of peripheral blood vessels, increased coronary flow and enhanced capillary permeability were observed on administration of these protein fragments [8]. In the early sixties the nonapeptide bradykinin and its precursors, kallidin and methionyl-kalUdin were isolated in pure form and their amino acid sequences determined soon after. 

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