Human serum fractionated

An affinity sorbent based on WPA-PG carrying immobilized human IgG was applied to the isolation of the first component of the complement (Cl) from human serum and for its separation into subcomponents Clr, Cls and Clq by the one-step procedure [126,127]. Cl was quantitatively bound to the sorbent at 0 °C. The activities of subcomponents Clq and Clr2r2 in the unbound part of the serum were found to be 0.8% and 3.3% of the initial activities in serum. This fraction, therefore, could be used as a R1 reagent for determining the hemolytic activity of Cl. Apparently, the neighboring macromolecules of immobilized IgG resemble to some extent an immune complex, whereas Cl formation is facilitated due to the mobility of polymer chains with the attached IgG macromolecules (Cl is usually dissociated in serum by 30%). After activation of bound Cl by heating (30 °C, 40 min) the activated subcomponent Clr is eluted from the sorbent. Stepwise elution with 0.05 mol/1 EDTA at pH 7.4 or with 0.05 mol/1 EDTA + 1 mol/1 NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent Cls and subcomponent Clq. 

Anthocyanins are poorly absorbed from the gastrointestinal tract and the mechanisms involved remain unclear. These compounds are usually recovered in very small amounts in human serum after oral ingestion (less than 1% of the dose) or in the IN fraction after in vitro digestion (about 5%). ° Unlike other polyphenols, anthocyanins constitute an exception because intact glycosides are recovered in the body (without deglycosylation prior to absorption). - This may be explained by either the instability of the free aglycone form or by a specific mechanism of absorption for anthocyanins. 

Fractionation of Serum and Column Capacity. A Fractionation of human serum on the SW-3000 column is shown in Figure 3. 

Figure 3. Fractionation of human serum proteins on Spherogel TSKSW 3000, The conditions were as in Figure 1. The analyses were made using (A) a 50-fiL injection loop with an analytical flow cell (B) a 100-L loop with a semipreparative flow cell or (C,D) a 500-L loop with a preparative flow cell.

Figure 12.5 illustrates the problem of using human serum digest analysis. The digest was fractionated using a 2.1 x 150 mm C18 column at pH 10 (50 J,L serum... 

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

Calcitonin is a polypeptide hormone that (along with PTH and the vitamin D derivative, 1,25-dihydroxycholecalciferol) plays a central role in regulating serum ionized calcium (Ca2+) and inorganic phosphate (Pi) levels. The adult human body contains up to 2 kg of calcium, of which 98 per cent is present in the skeleton (i.e. bone). Up to 85 per cent of the 1 kg of phosphorus present in the body is also found in the skeleton (the so-called mineral fraction of bone is largely composed of Ca3(P04)2, which acts as a body reservoir for both calcium and phosphorus). Calcium concentrations in human serum approximate to 0.1 mg ml-1 and are regulated very tightly (serum phosphate levels are more variable). 

A report on the binding of the anesthetic propofol to human serum albumin and to plasma presents a dataset that challenges simple notions of equilibria [70]. The unbound fraction of propofol was found to increase sharply at low drug concentrations. The authors appear to have carefully eliminated possible artifacts. Explanations based on cooperative binding modes are discussed though no clear explanation emerges. 

There is no agreement on the exact magnitude of the ultrafiltrable or high-molecular-mass fraction of Ni(II) in human serum. Moreover, pronounced species variations in the proportions of ultrafiltrable and protein-bound serum nickel have been demonstrated [289],... 

Tg Polyclonal Tg-Ab Tg-fragments Autoantibodies Human serum IgG fraction was found to hydrolyse thyroglobulin (Tg). Unknown immunogen 3.9 X 1(T2 3.9 X 10 3 nr 5.7... 

Plasmid DNA (pUC18) Fab fragment from an IgG purified from human sera pH 7.5,30°C Nicked DNA Autoantibodies Human serum IgG fraction (Fab) was found to hydrolyse DNA. Unknown immunogen 4.3 x 101 1.4 x 101 nr 6.5... 

Fig. 2.24. C30 chromatograms of carotenoids extracted from human serum (a) xanthophylls fraction, 7 93 (v/v) MTBE-methanol mobile phase (b) a- and / -carotenes fraction, 11 89 (v/v) MTBE-methanol mobile phase (c) lycopene fraction, 38 62 (v/v) MTBE-methanol mobile phase. Tentative peak identifications (a) 1, 13-c/s-lu- lutein 2, 13 r/.vlutein 3, a//-/ra s-lutein 4, zeaan-thin 5-7, unidentified P,e-carotenoids and 8, / -cyrptoanthin (b) 1-2, unidentified ae-carotene isomers 3, 15-eH -/f-carotenc 4, 13-cw-/ -carotene 5, all-trans-a-carotene 6, all-trans-P-carotene and 7, 9-ci.v-/3-carotene and (c) 1-11 and 13, c/s-lycopene isomers and 12, all-trans-lycopene. Reprinted with permission from C. Emenhiser el al. [51].

Fio. 1. Fraction of human serum liigh-density lipoprotein apoprotein (apo HDD, scheme 1. As in scheme 2, the method makes use of the combination of gel filtration and ion-exchange chromatography. Fraction IV is obtained in its dimer form. 

Fia. 2. Fractionation of human serum high-density lipoprotein ap>oprotein (apo HDD, scheme 2. Such a procedure takes advantage of the dimer— monomer conversion of fraction IV induced by the cleavage of the single disulfide bridge. R-IV = reduced fraction IV 8ME — 3-meroaptoethanol. Peaks — fraction HI = fraction IV fraction V. 

Besides fractions III, IV, and V, two other fractions, I and II have been described in preparations of human serum apo HDL, and HDL3. Although they are generally considered to be aggregates of the other fractions discussed above, these high-molecular-weight components have not yet been thoroughly studied. Such work is currently under way in this laboratory. 

Fig. 4. Amino add sequence of human serum high-density lipoprotein apoprotein (apo HDL), fraction IV (dimer). Data from Brewer et al. (B5).

A6. Albers, J. J., and Scanu, A. M., Isoelectric fractionation and characterization of polypeptides from human serum very low density lipoproteins. Biochim. Biophys. Acta 236, 29-37 (1971). 

El. Edelstein, C., Lim, C. T., and Scanu, A. M., On the subunit structure of the protein of human serum high density lipoprotein. I. A study of its major polypeptide component (Sephadex, fraction III). J. Biol. Chem. 247, 5842-5849 (1972). 

Scanu, A. M., Toth, J., Edelstein, C., Koga, J., and Stiller, E., Fractionation of human serum high density lipoproteins in urea solutions. Evidence for pol rpeptide heterogeneity. Biochemistry 8, 3309-3316 (1969). 

Most frequently, binding protein is added to the incubation mixtures as either serum or purified serum albumin. With human serum albumin, at equilibrium, the acceptor substrate will largely be protein-bound, when the bilirubin albumin molecular ratio is smaller than one (the dissociation constant of the first binding site of purified human serum albumin is approximately 7 X 10 M with 2 X 10 M for two additional binding sites) (J2). The first binding site of albumin, measured with rat serum, has a dissociation constant of about 5 X 10" M (M8). The unbound fraction will normally not exceed the very low solubility of the pigment. Addition of albumin to an alkaline solution of bilirubin, or its addition immediately after neutralization, prevents colloid formation, if the bilirubin albumin molecular ratio is smaller than one (B25). However, colloidal bilirubin, once formed, cannot be redissolved by the addition of albumin (B26). 

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