AASCIT Journal of Bioscience 2016; 2(6): 47-51 http://www.aascit.org/journal/bioscience ISSN: 2381-1250 (Print); ISSN: 2381-1269 (Online) Capturing the Antioxidant Polypeptides by Immobilized Hemin from Soybean and Ginkgo Xuluan Lin, Anjun Wang, Tingting Wang, Renqiang Li * Department of Biotechnology, Jinan University, Guangzhou, China Email address trqli@jnu.edu.cn (Renqiang Li) * Corresponding author Keywords Immobilized Hemin, Antioxidant Polypeptide, Affinity Chromatography Column, Soybean, Ginkgo Received: September 18, 2016 Accepted: October 16, 2016 Published: October 29, 2016 Citation Xuluan Lin, Anjun Wang, Tingting Wang, Renqiang Li. Capturing the Antioxidant Polypeptides by Immobilized Hemin from Soybean and Ginkgo. AASCIT Journal of Bioscience. Vol. 2, No. 6, 2016, pp. 47-51. Abstract After Sepharose 4B polymerbeads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column, which was used to capture the polypeptides from soybean and ginkgo. Equilibrated with deionized water and eluated with ph 3.6, 0.2 mol/l NaAc HAc buffer, those proteins obtained from this columne, including at least six polypeptides in soybean and two polypeptides in ginkgo, were demonstrated to possess high antioxidative activity (AA). AA of crude proteins of soybean was 10.52%, after purification, AA of the captured polypeptides by hemin was 28.72%. For ginkgo, AA of crude proteins and purified polypeptides was 7.72% and 18.45% respectively. This study offered a novel, rapid and effective method for preparation of antioxidative polypeptides from soybean and ginkgo. 1. Introduction Many natural foods, especially the plant foods, possess antioxidative activity (AA), because they contain large amounts of antioxidants. The antioxidants in many plants are proteins that have important function for the inhibition of lipid peroxidation, removal of excessive free radicals in vivo, and so on. Studies have showed that many proteins in plants, including rapeseed protein [1], soybean protein [2], ginkgo protein [3], have AA. Therefore, how to extract and purify these proteins with AA from the samples becomes an important part of the study. Although there are many different approaches that have been used[4-6], they are in general complicated or costly. Redox is an electron transfer process. The AA of the proteins may be achieved by electronic transfer. Heme is a very lively coenzyme and can bind with a lot of polypeptides to form the heme-containing proteins in organisms such as hemoglobins, myoglobin, cytochrome c, peroxidase including multi-heme cytochromes [7] and a series of heme-containing oxygenases [8]. The iron atom in heme, also known as iron porphyrin [9], is very active and can react with other materials through electronic transfer. Those polypeptides that could react with heme may be the proteins to possess AA. So it is worth trying to immobilize heme as a ligand to capture the antioxidative proteins. To immobilize hemin to make an affinity chromatography column, Sepharose 4B is a good candidate as polymer beads to bind with hemin because Sepharose 4B is active with its hydroxyl group after it was activated by epichlorohydrin, which would be helpful for the immobilization of hemin on Sepharose 4B beads. The purpose of this study is to explore a simple, fast and effective way to extract antioxidative polypeptides from soybean and ginkgo.
48 Xuluan Lin et al.: Capturing the Antioxidant Polypeptides by Immobilized Hemin from Soybean and Ginkgo 2. Materials and Methods 2.1. Materials Soybean and ginkgo were purchased from the market; Sepharose 4B was from Amersham. Hemin was from Sigma. Linoleic acid, potassium thiocyanate, EDTA, epichlorohydrin, 1,4-dioxane and all other chemicals used in SDS-PAGE or for buffers were of analytical or chemical grade and were purchased from Guangzhou Chemical Co.Ltd. Markers for SDS-PAGE were supplied by Shanghai Sheng Zheng Biotechnology Co.Ltd. 2.2. Preparation of Immobilized Hemin Column Activation of Sepharose 4B: Twenty-five grams of wet Sepharose 4B was washed using 1 mol/l NaCl and deionized water at Buchner s funnel. These polymer beads were mixed with 19 ml 2 mol/l NaOH, 5ml epichlorohydrin and 25 ml 56% 1,4-dioxane, shaken for 2 h at 40 C [10]. After being washed at funnel by using deionized water, Sepharose 4B beads were saturated in 0.1 mol/l, ph 9.5 Na 2 CO 3 NaHCO 3 buffer. Combination of hemin with Sepharose 4B: Hemin was first dissolved in small quantities of 15% ammonia water and then in deionized water to make a 2 mmol/l solution, of which 29 ml was mixed with activated Sepharose 4B beads. The mixture was shaken at 40 C for about 24 h and washed at funnel using deionized water to remove the hemins that were not attached to polymer beads. Coupling rate between hemin and the beads was expressed using mg hemin fixed per gram of wet Sepharose 4B beads. An affinity chromatography column with 11 cm 1.5 cm was made using the hemin as a ligand. For comparison, a control column of Sepharose 4B beads without hemin coupled was also made. 2.3. Preparation of the Protein Crude Extracts Hundred and twenty grams of washed and peeled ginkgo or 100 g of soybean immersed in water for 6 h were respectively bled in cold phosphate buffer (0.01 mol/l, ph 7.2). The homogenate was extracted with stirring slowly using deionized water overnight at 4 C and then centrifugated (4 C, 10000 rpm, 30 min) to collect the supernatant. The centrifugation was done for three times to remove the yellow oil slick in the supernatant. The supernatant was salted out with 80 percent of ammonium sulfate. The precipitation was collected by centrifugation (4 C, 10000 rpm, 15 min) and dissolved in PBS, then dialyzed against deionized water. The protein crude extracts of ginkgo or soybean was obtained after centrifugating the dialysate. 2.4. Sample Loaded and Eluted New black immobilized hemin column and the control column were washed fully by using 0.1 mol/l ph 9.5 Na 2 CO 3 NaHCO 3 buffer, deionized water and 0.2 mol/l, ph 3.6 NaAc HAc buffer, respectively, and then equilibrated with deionized water. Ten milliliters of protein crude extracts of soybean and ginkgo were respectively loaded to immobilized hemin affinity chromatography column at a flow rate of 0.2 ml/min. After being washed fully with deionized water, the eluant 0.2 mol/l, ph 3.6 NaAc HAc buffer were used to elute the column and the peak fraction at O.D. 280 were collected. 2.5. SDS-PAGE The eluate proteins from the immobilized hemin column were concentrated by vacuum cold drying apparatus (Heto, High Technology of Scandinavia, Denmark) and dialyzed against deionized water before mixing with 1% SDS-mercaptoethanol solution as the SDS-PAGE sample. Gel concentration used in SDS-PAGE was 13%. Analysis of SDS-PAGE pattern was performed using Electrophoresis Image Analysis System (FR-980, Furi Company, Shanghai, China). 2.6. Determination of Antioxidative Activity of Proteins Antioxidative activity of protein samples were measured with linoleic acid-potassium thiocyanate method [11]. 1.5 ml of 0.15 mol/l, ph 7.0 phosphate buffer was mixed with 100 µl of protein sample in 5 ml tube, then 100 µl of ethanol containing 50 mmol/l linoleic acid and 25 µl of 50 mmol/l FeCl 2 -EDTA solution was respectively added to this tube. The tube was covered with the stopper and treated in water bath in dark at 50 C for 4 h. As a control, the same operation was performed by replacing the protein samples with distilled water. 100 µl of the above mixture that had reacted for 4 h was mixed with 3 ml of 75% ethanol, 100 µl of 1.0 mol/l HCl containing 1 mol/l FeCl 2 and 100 µl of 30% potassium thiocyanate for 3 min reaction. The O.D. was measured at 480 nm to obtain absorbance As (sample) and A 0 (control). Measurements were performed repeatedly, and averaged. Antioxidative activity (AA) was got by calculation according to AA = (A 0 -As)/A 0 100%. 3. Results and Discussion 3.1. Separation of the Column As expected, the immobilized hemin column was obtained and the coupling rate was very hight with more than 0.122 mg hemin fixed by gram of wet Sepharose 4B beads. Fig. 1 and 2 showed respectively the separation of soybean and ginkgo proteins from the hemin-sepharose 4B column. Whether soybean or ginkgo, most of the proteins were not adsorbed by the column to become flow-through peak (large peak), only a small portion of the proteins was adsorbed by the affinity column, named as elution peak (small peaks), which could be considered to have interaction with hemin. In
AASCIT Journal of Bioscience 2016; 2(6): 47-51 49 the control column, no elution peak was appeared both soybean and ginkgo, which demonstrated that the eluate proteins were captured by the immobilized hemin. Big peak (1): flow-through fraction; small peak (2): elution fraction. Equilibrium solution: deionized water; eluent: 0.2 mol/l,ph 3.6 NaAc-HAc buffer. Fig. 1. Elution profile of soybean proteins from hemin-sepharose 4B affinity chromatography. Big peak (1): flow-through fraction; small peak (2): elution fraction. Equilibrium solution: deionized water; eluent: 0.2 mol/l,ph 3.6 NaAc-HAc buffer. Fig. 2. Elution profile of ginkgo proteins from hemin-sepharose 4B affinity chromatography. 3.2. SDS-PAGE The SDS-PAGE pattern of eluate proteins from immobilized hemin column was showed in Fig. 3. Most proteins were not adsorbed by the hemin both soybean and ginkgo, but there were at least six polypeptides in soybean proteins that could be captured by the hemin, and two polypeptides could react with hemin in ginkgo proteins, which indicated that the immobilized hemin could selectively capture related proteins.
50 Xuluan Lin et al.: Capturing the Antioxidant Polypeptides by Immobilized Hemin from Soybean and Ginkgo was considered relating with the sulfhydryl (-SH) and composition of amino acids in molecular structure of the protein [12], the R groups of these amino acids could transfer electrons when they worked in biochemical process, so these proteins possessed AA and could react with active hemin, which was why the hemin as ligands could adsorb the antioxidative proteins. From Fig. 3, the molecular mass of a main polypeptide captured by the hemin in soybean and ginkgo was all about 20.1KDa, which maybe give out some information of their relationship in molecular structure, biofunction or evolution. In depth study on these polypeptides would be helpful for understand the structural characteristics and mechanism of antioxidative proteins. 4. Conclusions Lane a: flow-through proteins of ginkgo; lane b: eluate of ginkgo; lane c: eluate of soybean; lane d: flow-through proteins of soybean; lane M: markers. Fig. 3. SDS-PAGE pattern of proteins captured by hemin-sepharose 4B column. 3.3. AA of Captured Proteins The protein crude extracts, flow-through proteins and captured proteins of soybean and ginkgo were first concentrated respectively, and then diluted to a same concentration of 1.95 mg/ml. They were measured for their AA, which was showed in Table 1. Both soybean and ginkgo, the whole proteins possessed certain AA. When passed through the immobilized hemin column, those proteins with AA were adsorbed by the column. The flow-through proteins almost had not the AA. The elution proteins or captured proteins possessed higher AA with more than two times compared to the whole proteins, which was obviously due to the purification and concentration of antioxidative proteins. These results demonstrated that the immobilized hemin could capture well those antioxidative polypeptides. Samples Soybean Ginkgo Table 1. Antioxidative activity of different protein samples (n = 3). Antioxidant activity (AA%) the whole proteins 10.52 flow-through proteins 3.29 eluted proteins 28.72 the whole proteins 7.72 flow-through proteins -0.99 eluted proteins 18.45 For soybean, the proteins passed through the column also retained some AA as shown in Table 1. This phenomenon explained that in practice it was not all antioxidative proteins that could be captured by the hemin. Fig. 1, 2 and 3 indicated that the adsorbed proteins by the column were only a small part in whole proteins of both soybean and ginkgo, but these parts adsorbed by the column had a higher AA, which indicated that heme as ligands could selectively adsorbed those proteins with AA. AA of proteins An immobilized hemin affinity chromatography column was used to separate antioxidative proteins from soybean and ginkgo. There were at least six polypeptides in soybean and two polypeptides in ginkgo that could be captured by the hemin. Those proteins obtained from the column were demonstrated to possess higher AA with more than two times compared to the whole proteins, which was obviously due to the purification and concentration of antioxidative proteins. In soybean, AA of crude proteins and the captured polypeptides by hemin was 10.52% and 28.72% respectively. For ginkgo, AA of crude proteins and purified polypeptides was 7.72% and 18.45% respectively. This study offered a novel, rapid and effective method for preparation of antioxidative polypeptides from soybean and ginkgo. References [1] Nichole C., Ying Z., Marian N. et al.,(2008), Antioxident activity and water-holding capacity of canola protein hydrolysates. Food Chemistry, 109 (1): 144-148. [2] Mesa M. D., Silvan J. M., Olza J. et al., (2008), Antioxidant properties of soy protein-fructooligosaccharide glycation system and its hydrolyzates. Food Research International, 41 (6): 605-615. [3] Lena M. G., Philip J. B., (2002), Antioxidant capacity in Ginkgo biloba. Food Research International, 35 (9): 815-820. [4] Zhang LL., Yan QF., Wang T., (2007), Study on isolation and antivity of bioactive peptides. Food Science, 28 (5): 208-210. (in Chinese) [5] Ren HW., Wang CQ., Song YX., (2009), Study on the isolation and antioxidant of black-soybean peptides. Nat Prod Res Dev, 21: 136-139. (in Chinese) [6] Chen TF., Huang YC., (2006), Antioxidant and anticancer activities of selenium-containing phycocanin purified from spirulina platensis. Academic Periodical of Farm Products Processing, 8: 55-58. [7] Mowat CG., Chapman SK., (2005), Multi-heme cytochromes--new structures, new chemistry. Dalton Trans, 7 (21): 3381-3389.
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