Equilibration, sample, and washings with Buffers A and B were carried out as indicated in Section 4.1, Section 4.2.1, and Section 4.3. Next, 10 CV of linear gradient 5 mM to 25 mM CaCl2 (in Buffer B) and 5 CV stepwise Buffer B + 25 mM CaCl2 were carried out. They were then washed with Buffer B (200B) and the elution with Buffer C was performed as in Section 4.2.1. Proteins identified by mass spectrometry of the fraction “25” from plasma purification on ANX Sepharose FF using stepwise 10 mM and 25 mM CaCl2 gradient.
- It was possible to observe high molecular mass proteins present in all collected fractions (Figure 7).
- The matrix of Sepharose resins is agarose and the matrix of the Fractogel resins is metacrylate.
- A second column was needed to separate FVIII from PCC proteins, and it was successfully achieved by immobilized metal affinity chromatography 16,18.
- These results indicate that the CaCl2 gradient step did not have a negative affect on FVIII activity and significantly improved the purification factor.
- To identify the proteins that eluted with the increase of calcium concentration on ANX Sepharose FF purifications, we analyzed the bands of the SDS PAGE reducing gel of six different fractions eluted by a 5 to 25 mM calcium gradient using mass spectrometry.
They review the developments in their agricultural statistics systems since the last session and exchange ideas with experts from FAO and other organizations on the state of food and agricultural statistics in the region. FAO uses this occasion to inform the member countries about its activities during the preceding biennium, particularly in Asia and the Pacific region. The protein contents of the investigated polysaccharide preparations were determined adopting the method of Lowry et al. 22 and using bovine albumin as a standard. Molar sugar content, as detected by HPLC, of the biologically active fractions along with the hydrolysate and the mother algal extract.
These results indicate that the vitamin K dependent coagulation factors bind stronger on Fractogel EMD DEAE than on TMAE. Anion exchange resins are widely used in the plasma fractionation processes 3,12,13,14,15 due to their high capacity, good yields and low cost compared to other chromatographic techniques. Upon loading whole plasma directly onto an anion exchange column, without the cryoprecipitation step, FVIII and PCC are strongly adsorbed and cannot be eluted separately using NaCl gradient 16,17. A second column was needed to separate FVIII from PCC proteins, and it was successfully achieved by immobilized metal affinity chromatography 16,18. Conventional affinity chromatography involves the immobilization of a ligand for which the protein of interest has an affinity. Pseudoaffinity elution in ion exchange chromatography differs from affinity chromatography in that there is no ligand immobilized on the matrix for which the protein of interest has affinity, and this matrix can be a conventional ion exchange resin or hydrophobic interaction 19.
Our results indicate that prothrombin complex could be eluted with CaCl2 without affecting FVIII activity using all five tested resins. Fourier transform infrared spectroscopy was performed for S1, V45, and all 8 column fractions. Samples were mixed with potassium bromide in the form of 1-mm pellets and analyzed in a TGA/FT-IR Nicolet 380 spectrometer for a range of wavenumbers between 500 and 4000 cm−1.
5.2. Fractogel EMD TMAE Chromatography with Ca2+ 5 mM to 50 mM Linear Gradient
- Therefore, for the elution of vitamin K dependent proteins, a calcium gradient was introduced after the wash of the unbound proteins.
- The flow rate was 15 mL min−1 during plasma application and 25 mL min−1 during other purification steps.
- Green Ulva lactuca algae were collected in summer time from the Mediterranean Sea in Egypt, specifically Alexandria in Abou Kir region (N 31°19′ E 30°03′).
- The citrate buffer is used in FVIII purifications because of its anticoagulant activity, but it is also able to chelate Ca2+ ions.
With both columns it was possible to establish a stepwise protocol to elute FVIII and PCC (FIX) separately. Noteworthy is that it was possible to elute FVIII from the Q Sepharose FF column by increasing CaCl2 concentration to 65 mM from 25 mM citrate buffer, pH 6.0 containing NaCl 200 mM without formation of coagulum (Figure 4a, lane 65A). However, the FVIII yield obtained in this purification was comparable to that obtained with the FVIII eluted with 500 mM NaCl 20 and the purification factor was comparable to that observed with all other resins tested in this work employing the CaCl2 gradient step (Table 1).
This is probably indicative of a higher degree of cooperativity in F5 treatments when compared to all others. An explanation for such a trend could most likely be due to the presence of multi-subunit «active components.» Treatment of MCF7 cells with F5, however, resulted in a significantly lower Hill coefficient when compared to the treatment of either HePG2 or A549 cells. This suggests that ligands in the same fraction possibly interact with different sets of target molecules from one cell line to another.
Another fraction that showed no antitumor activity, even though it comprises the same main functional groups present in fractions F4 and F5 (hydroxyl, amide, sulfate, and thiocarbonyl), is F3. This could be ascribed to its possession of unequal ratio of sulfate to carbohydrate content and probably due to its lower DP as compared to F4 and F5. However, F3 possesses an additional phenolic group that could have possibly played a role in imparting an antioxidant activity for this fraction that is comparable to those of F4 and F5. Antioxidant activity has been reported in the literature to be directly related to the phenolic content 21,36. 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. As can be seen in Table 3, four fractions S1, F4, F5, and F8 showed % lethality above 75.
SDS-PAGE 7.5% analysis under non-reducing conditions of the purification of plasma on ANX Sepharose FF with CaCl2 5 to 25 mM linear gradient in 25 mM citrate, 200 mM NaCl, pH 6.0 buffer. Interestingly, F6 and F7 showed no antitumor activity and relatively low antioxidant activity relative to F4 and F5, although they share the same DP of 3 with these two fractions. This could be attributed to their low protein content and hence their possession of less functional amide groups as compared to fractions 4 and 5. This is in addition to their highest sulfate contents amongst other fractions, which could have affected their antitumor activity as was the case with their antioxidant activity.
Materials and Methods
The open source reference implementation of CryptoNote was coded from scratch based on the CryptoNote reference implementation, and is not a inf8 exchange fork of Bitcoin. Infinium-8 aims to be a fungible and untraceable digital medium of exchange. The volume of the Fractogel EMD DEAE column was 24 mL and flow rates were as indicated in Section 4.5. The resin was regenerated by sequentially washing with 2 CV 0.5M NaOH (with 1 h pause), 5 CV 2M NaCl and 10 CV purified water and stored in 10 mM NaOH. Equilibration, sample, and washings with Buffers A and B were carried out according to Section 4.1, Section 4.2.1 and Section 4.4. The KαL 1 /KαL 0 intensity ratio of fluorine is measured in five fluorine compounds with a crystal spectrometer.
3.3. ANX Sepharose FF Chromatography with Ca2+ Stepwise Elution
All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells were suspended in Roswell Park Memorial Institute RPMI 1640 medium for HePG2, MCF7, and HCT116, and in Dulbecco’s Modified Eagle Medium DMEM for A549. Where Q is the absorbance ratio (A1/A2), and A1 and A2 are the respective absorbances of SPs samples before and after reduction. In the past decades, marine seaweeds have gained much interest as wealthy resources of bioactive compounds.
In addition, this study elucidated some of the important factors that could influence the bioactivity. Major factors that, to our best knowledge, have not been reported elsewhere are the carbohydrate to protein to sulfate composition and the sugar composition. In view of these influential factors, tailor-made oligosaccharides could be synthesized and designed as food supplements.
The above findings are in agreement with the antitumor results shown earlier, where F5 exhibited the highest percentage lethality on HePG2. Elution was carried out with a 5 CV linear gradient of 100 mM to 600 mM NaCl in Buffer A and 5 CV step of the Buffer A + 600mM NaCl. Ulva lactuca, or as commonly known by “sea lettuce”, is an edible green seaweed from which the SPs, ulvans, are extracted. Their molecular weight ranges from 189 to 8200 KDa and they are composed of units of mono or disaccharides such as rhamnose, xylose, and iduronic or glucuronic acid. The most abundant unit is ulvan biouronic acid with sulfate at C3 where the acid unit could be either glucuronic or iduronic acid 2. Twenty CV of FFP were applied to the column and the unbound proteins were washed with 14 CV of Buffer A. FT was collected in fractions of 2 CV.
Figure 9 shows that the 3 non-vitamin K dependent proteins (C4bBP, C4 and Fibrinogen), identified by mass spectrometry, are the contaminant proteins present in the obtained PCC. Considering the SDS-PAGE analyses shown in Figure 3, bands of these proteins are present in other, if not all, gels. It seems that these proteins are present in the collected fractions of all resins and in the FVIII containing fractions.
