It circulates in the bloodstream complexed with von Willebrand factor, which acts as a transporter 3 and prevents the degradation of FVIII by the action of serine proteases, including activated Protein C and activated FX 4. FVIII is a high molecular mass (300 kDa) and labile protein present in very low concentrations in plasma (150 ng mL−1) 4,5. The hydrolysate (V45) was further purified using diethylaminoethyl (DEAE) cellulose anion-exchange column chromatography.
2. Chromatography of Plasma on Q Sepharose FF
- In addition to sugar composition, the complexity of the polysaccharide in terms of the variety of sugar units constituting its chain is another important influencing factor.
- As a preliminary investigation, the lethality percentage for each algal fraction on each of four cancer cell lines was determined to test its antitumor activity.
- By means of this facile process, products with enhanced biological activities could be obtained in a few number of steps and via a green approach with no solvent requirements.
- No general protocol could be established based on the strength of the anion exchanger nor the chemical nature of the matrix.
- Nine lanes of the gel were loaded with the fraction “25” in a non-reducing polyacrylamide gel 6% (Figure 9b).
- The same finding was reported in a study on Ganoderma atrum mushrooms 19, and in another study performed on polysaccharides extracted from the brown seaweed Saragasum pallidum.
They gave one short and wide peak in the calcium gradient step, except for the purification on ANX Sepharose FF, which presented two peaks. The first peak eluted with CaCl2 10 mM and the second peak, which contained the vitamin K dependent proteins, eluted with 25 mM CaCl2 (Figure 4f). The same bands that eluted with 10 mM CaCl2 are present in the purifications by all tested resins, but only in the purification by ANX Sepharose FF part of these proteins eluted separately.
The flow rate was 15 mL min−1 during plasma application and 25 mL min−1 during other purification steps. Where A0 is the absorbance of the sample-free DPPH solution, A1 is the absorbance of the tested samples in absence of DPPH, and A2 is the absorbance of the tested samples in presence of DPPH. IC50 was estimated as the sample concentration (μg/mL) required to scavenge 50% of the DPPH. The sulfate content, on the other hand, was determined after cleavage of the polysaccharide molecule using the method of Larsen et al. 48. Sulfate contents of the hydrolysates were determined to adopt the barium chloride turbidimetric assay of Garrido 49, with some modifications 21. The amount of sulfate was estimated based on the anhydrous sodium sulfate standard calibration curve.
- The antitumor cell test depends basically on the cytotoxic effect of each of the samples on the cancer cells.
- The higher molecular mass proteins observed on the Q Sepharose FF gel (Figure 3a), due to the higher CaCl2 concentration used (65 mM), were not observed on ANX Sepharose FF purification gel where the highest CaCl2 concentration achieved was 25 mM.
- The fractions F5 and F8 also showed comparable antioxidant activities but at lower concentrations.
- The fraction of the non-adsorbed proteins, which can be called the clotting factor-poor fraction, containing roughly 99% of the plasma proteins including albumin, immunoglobulins and other proteins, can continue in the plasma fractionation process.
- The ANX Sepharose FF resin used was 51 mL and the used flow rates were as indicated in Section 4.2.
- The study also looks into the parameters that might affect the antioxidant and antitumor activities of these fractions.
Table 4.
Proteins identified by mass spectrometry of the fraction “25A” from plasma purification on ANX Sepharose FF using stepwise 25 mM CaCl2 gradient. Hill coefficients (Table 5) were determined for fractions that showed lethality ≥75% and were all above 1. Hill coefficients can be used to give an idea of the degree of cooperativity among ligands as they bind to target macromolecules. Coefficients above, below, and equal to 1, respectively, indicate positive, negative, and non-cooperative ligand binding 37. This suggests that the binding of one ligand promotes the binding of other ligands for fractions S1, F4, F5, and F8. The highest Hill coefficients were observed in treatments of fraction F5; treatment of F5 on HepG2 cells showed the greatest Hill coefficient (2.591 ± 0.155) followed by that on A549 (2.512 ± 0.156) and MCF7 (1.780 ± 0.097) cells, respectively (Figure 5).
In particular, sulfated polysaccharides (SPs) extracted from algal species have shown various biological and physiological activities. Conceptualization, E.C.; Investigation, G.P.F., S.H.A., V.W.N. and J.R.D.S.; Formal analysis, G.P.F. and M.P.B.; Funding acquisition, E.C. And E.A.L.M.; Writing, E.C. All authors have read and agreed to the published version of the manuscript. The volume of DEAE Sepharose FF column was 28 mL and the used flow rates were as indicated in Section 4.2. The ANX Sepharose FF resin used was 51 mL and the used flow rates were as indicated in Section 4.2. Resin was regenerated by sequentially washing with 2 CV 1M NaOH (with 1 h pause), 5 CV 2M NaCl and 5 CV purified water and stored in 10 mM NaOH.
Table 5.
SPs inf8 exchange extracted from Ulva intestinalis and fractionated onto a silica–silica column demonstrated antioxidant in addition to immunomodulatory activities since they stimulated pro-inflammatory cytokines production 11. Ulvans from Vietnamese Ulva lactuca were also shown to be cytotoxic to hepatocellular carcinoma, in addition to human breast and cervical cancer cell lines 16. Interestingly, the citrate buffer played a significant role in the purification profile. The citrate buffer is used in FVIII purifications because of its anticoagulant activity, but it is also able to chelate Ca2+ ions. Bis-tris and MES buffers were chosen for comparison because they do not chelate calcium ions (or very weakly), have a pKa near 6 (6,46 and 6,15, respectively), and have completely different chemical structures (amine and sulfonic acid, respectively). Compared with the citrate buffer, using Bis-tris and MES buffers, the same 200A peak was not observed (Figure 1c,f,g), and separation of FVIII and FIX was not successful, because 20 to 30% of PCC (FIX) did not elute with CaCl2 25 mM (Figure 2c,f,g).
An anomalous reduction of this intensity ratio was observed in KF and SrF 2 , which is attributed to resonance electron transfer from the metal ion to the spectator vacancy in the fluorine ion. The measured relative intensities are compared with the theoretical estimates of Aberg. Finally, the Hill slope was defined as the steepness of a given dose response curve and was considered to be equivalent to the Hill coefficient 57.
Figure 8.
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Spots from fractions F2, F9, F12 and F15, which were taken from the reducing SDS-PAGE (7.5% acrylamide,) are shown in Figure 8a, which is the same gel shown in Figure 3c. Spots from fractions F4 and F9 were taken from 5 to 15% acrylamide reducing SDS-PAGE (Figure 8b). The identified proteins were Complement C4 binding protein, Fibrinogen, Complement C4 and Prothrombin. In a typical fractionation scheme, plasma bags are thawed at 1 to 4 °C and cryoprecipitate is isolated by refrigerated centrifugation, providing concentrates of FVIII, von Willebrand factor and fibrinogen 8. The vitamin K dependent proteins remain in the supernatant (called cryo-poor plasma), along with albumin, IgG and other proteins.
Activities of FVIII, Protein C, FIX and Prothrombin were evaluated using the chromogenic method in microplates, as recommended by the manufacturer. Samples of the FFP pool were used to build the calibration curve for each experiment. Considering that, by definition, one unit of FVIII is the amount of FVIII activity in one mL of normal plasma, the FVIII activity was expressed in arbitrary unit (U), which was used to calculate the percentage of the recovered activity and the specific activity.