RFC 3711 The Secure Real-time Transport Protocol SRTP

The absence of the peak 500 in Figure 4a and the similarity of the protein profile lane 65A of Figure 4a (purification on Q resin) and lane “500” of Figure 4d,f (purification on ANX) indicate that FVIII eluted due to increase of the ionic strength. Furthermore, work with highly concentrated Ca2+ solutions is not recommended because of the known property of Ca2+ ions to promote coagulation. For the purifications on the Fractogel EMD, TMAE, and DEAE resins, the CaCl2 gradient step was carried out immediately after the reequilibration wash of the unbound proteins.

2. Enzymatic Hydrolysis and Anion-Exchange Chromatography

Fractions S1, F4, and F8 were lethal to HCT116, HePG2, and MCF7 cancer cells, respectively; whereas F5 was almost 100% lethal to both HePG2 and MCF7, and 80% lethal to A549 cancer cells. The mechanism of DPPH scavenging activity is that of hydrogen transfer from the antioxidant to the DPP hydrazyl (radical) to convert it into DPP hydrazine. This is to avoid the presence of the active free radicals, which have the capability of degenerating the proteins, lipids, and DNA in human bodies or foods on the shelf, and, hence, lead to degenerative diseases 33. The hydrogen transfer is suggested to take place through a possible reaction between the radical and amine or amide groups present in the antioxidant 34. As deduced from the FTIR analysis, the amide group is shown in all fractions and, therefore, all of them have antioxidant activity. The highest in protein content are F2 and F4 and they are the only two fractions that showed antioxidant activity above the 60% so it could be attributed to the higher amount of protein, which indicates higher content of amide groups.

Dose–response curves (Figure 3) were generated for these fractions (S1, F4, F5, and F8) and used to determine LC50, LC0, and Hill coefficient values. LC50 and LC90 represent the concentration of the extract or fractions that led to death of the cancer cells by up to 50% and 90%, respectively. Equilibration, sample, and washings with 10 CV of Buffer A, according to Section 4.1 and Section 4.2, and 10 CV of Buffer B (25 mM sodium citrate, 200 mM NaCl and 5 mM CaCl2, pH 6.0) (200A) were carried out. Next, 10 CV of 5 mM to 100 mM CaCl2 (in Buffer B) linear gradient and 5 CV stepwise Buffer B + 100 mM CaCl2 were undertaken. After a second wash with 5 CV of Buffer B (200B), elution with 4 CV of Buffer C (25 mM sodium citrate, 500 mM NaCl and 5 mM CaCl2, pH 6.0) was performed. In all experiments, five bags of FFP were thawed in a water bath at 37 °C, and the pH of the pool was adjusted to pH 6.0 with 0.2 M citric acid and directly applied to resins.

Figure 4.

In this experiment it was possible to determine the activity of Protein C, since the activity in the fractions collected in the CaCl2 linear gradient were lower than the detection limit of the chromogenic method. The yield of Protein C in this experiment was 44% and the purification factor was 126. It was also reported that column purification enhances the antioxidant activity of the extracted SPs. Polysaccharides extracted from Ganoderma atrum and purified onto a gel filtration chromatography column manifested potent antioxidant activities and the activity of one of them was comparable to that of ascorbic acid 19. Hot water polysaccharide extracts of Gracilaria rubra were purified onto anion exchange and gel filtration columns, and the purified fractions exhibited good antioxidant and immunostimulating activities 20. In our previous work 21, we reported the enzymatic hydrolysis of the red algal extracts of Pterocladia capillacea using different enzymes.

1. Molar sugar content, as detected by HPLC, of the biologically active fractions along with the hydrolysate and the mother algal extract.
2. All three of these processes have one thing in common, namely, they result in F K x rays.
3. FIX (Biophen Factor IX) and Prothrombin (Biophen Prothrombin) chromogenic assay kits were purchased from Hyphen Biomed (Neuville sur Oise, France).
4. Samples were analyzed for mono- and disaccharides by high-performance liquid chromatography (HPLC) according to AOAC 51.
5. 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.
6. They are frequently captured by anion exchange chromatography, resulting in a mixture of intermediate purity, called the prothrombin complex concentrate (PCC) 9,10.
7. The cancer cells used were all from the American type culture collection ATCC (USA) and were four types (HCT116, colon cell line; A549, lung carcinoma cell line; HePG2, human hepatocellular carcinoma cell line; MCF7, breast carcinoma).

7.1. Protein Quantification

All chromatographies were carried out using Äkta® Explorer or Purifier systems run with Unicorn 5.01 software (Cytiva). Compared with the traditional Cohn method, in the proposed purification method it is possible to obtain an enriched FVIII fraction without the need of cryoprecipitation, thus avoiding refrigerated thawing and centrifugation at low temperatures. Attempting to avoid cryoprecipitation is not new 24,25,26,27; however it was necessary to use 2 ion exchange columns to separate FVIII from PCC, one of them being used in batch mode, which makes it more difficult to scale up the process. Finally, the method we describe in this work can be easily integrated into any fractionation process. 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.

1. In addition, this study elucidated some of the important factors that could influence the bioactivity.
2. In this fraction, no prothrombin activity was found, and the FIX activity found was negligible (0.1%), indicating that the separation of FVIII from the prothrombin complex proteins succeeded.
3. 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.
4. The hydrogen transfer is suggested to take place through a possible reaction between the radical and amine or amide groups present in the antioxidant 34.
5. 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 0.2 M NaOH.
6. The Asia and Pacific Commission on Agricultural Statistics (APCAS) is a statutory body of FAO.

During the purification of plasma on DEAE Sepharose FF using the protocol without the CaCl2 gradient step, 35% of FVIII eluted in the 200 mM intermediate wash. Therefore, the calcium gradient was introduced after the wash of the unbound proteins. Several studies reported the inf8 exchange antioxidant activity of Ulva lactuca extracts 3,4,5,6,7,8,9. In addition to their potent antioxidant activities, ulvans have shown promising anticancer activities via one or more of the following mechanisms; anti-metastasis, anti-angiogenesis, immunity modulation, apoptosis, and antioxidant activity 10.

As can be seen from the figure, the antioxidant activity is concentration dependent for S1 and all column fractions. It is also clear from the figure that at all concentrations, the activity of the mother fraction (S1) decreased with enzymatic hydrolysis. Furthermore, the anion exchange purification of the enzymatically hydrolyzed fraction did not improve the activity. 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. In the latter, purification of the crude extracts onto DEAE column reduced their antioxidant activities 18. Finally, spots of the fraction that elutes with 25 mM CaCl2 from purification using two steps CaCl2 gradient (Figure 4f, lane “25”) were analyzed.

Furthermore, the band pertaining to the O–H bending vibration of phenolic groups appeared at 1365 cm−1 in F3 only, while that corresponding to the thiol stretching vibration (S–H) appeared only in F8 at 2583 cm−1 31,32. In conclusion, all fractions along with S1 and V45 contain hydroxyl, sulfate, and amide groups. All fractions together with S1 and V45 and apart from (F3–F7) contain sulfoxide groups. The main functional groups present in the analyzed fractions are summarized in Table 2.

2.1. Q Sepharose FF Chromatography with Ca2+ 5 mM to 100 mM Linear Gradient

Nine lanes of the gel were loaded with the fraction “25” in a non-reducing polyacrylamide gel 6% (Figure 9b). Bands shown in Figure 9b were excised horizontally and treated with trypsin, DTT and iodoacetamide according to the described method. Part of the prepared samples were directly analyzed by mass spectrometry and part was analyzed by RF-HPLC-C18 and then by mass spectrometry. In this experiment, two more vitamin K dependent proteins were identified, FIX and Protein Z (Table 4).

The recovery of Prothrombin and FIX was 100%, and the purification factors were 689 and 496, respectively. The yield of FVIII in the fraction “500” was 59%, with a purification factor of 220 (Table 1). The proposed process combining enzymatic hydrolysis and ion-exchange chromatography could potentially be used in food and pharmaceutical industries as a post extraction step to produce compounds with better bioactivities than the polysaccharide extracts. The process is relatively facile with few number of steps and is green with no solvent requirements.

This last protein could only be identified by the mass spectrometry of a compound eluted from the RP-HPLC C-18 analysis of the lowest molecular mass bands of the non-reducing gel shown in Figure 9b. Therefore, for the elution of vitamin K dependent proteins, a calcium gradient was introduced after the wash of the unbound proteins. We evaluated the elution of PCC by changing the Ca2+ concentration in the buffer solutions and then FVIII was eluted by high concentrations of NaCl. With the enzymatic hydrolysis of S1 to produce V45, the carbohydrate and sulfate contents decreased at the expense of the protein content. This is probably because Viscozyme broke down the saccharide linkages and, as a result of centrifugation and decantation, some of the broken soluble sugars were removed. Among the eight column fractions, F4–F8 have the highest sulfate contents, with a corresponding degree of polymerization of 3–5.