A Scientific Journal of Biological Sciences Comparative study of protein profile of eight benthic marine macroalgae by SDS PAGE S. Chakraborty 1 , S.C. Santra 2 and T. Bhattacharya 3 1 Dept. of Biological and Environmental Science, N.V.Patel College of Pure and Applied Sciences, V.V. Nagar, Gujarat-388120, INDIA 2 Department of Environmental Science, University of Kalyani, Nadia, West Bengal - 741235 3 P.G. Dept. of Environmental Science and Technology,Institute of Science and Technology for Advanced Studies & Research, (ISTAR),V. V. Nagar 388120, Gujarat, INDIA (Received 25 March 2010; accepted 28 April 2010) SDS PAGE was tested as an analytical tool for the isolation and identification of proteins from eight marine benthic macroalgae (Catenella, Polysiphonia and Gelediella of Rhodophyceae, Rhizoclonium, Enteromorpha, Lola and Ulva of Chlorophyceae and Dictyota belonging to Phaeophyceae,) thriving in the mangrove vegetation of Sunderban, India. Chracteristic protein banding pattern was observed for each genus. The pattern for Dictyota consists of 7 bands located between 29 and 205 kDa with the presence of triplicate bands at 43 kDa. The pattern for Gelediella consists of four bands between 43 and 205 kDa. Rhizoclonium consists of six bands lying between 3 and 205 kDa. Four bands between 3 to 43 kDa were characteristic of Lola. Catenella exhibited six bands within a wide range of 6.5 to 205 kDa. Polysiphonia consists of three bands within a wide range of 3kDa to 205kDa. Enteromorpha showed very interesting pattern of 4 clear bands, while Ulva showed nearly 7 bands. SDS PAGE appears to be suitable for the identification of benthic marine macro algae. Key Words: Bands, Gel electrophoresis, macroalgae, protein INTRODUCTION Innumerable works done on whole cell protein analysis of algae have revealed that they are rich in protein content and also potentially important for containing many bio-active substances which could be skillfully isolated and employed for various food, feed, pharmaceutical and cosmetic industry. Therefore protein characterization of marine algae is a major factor deciding the success of experiments in algal biochemistry. In some species like Palmaria palmate and Porphyra tenera proteins can represent 35 to 47% of the dry weight. Spirulina, a fresh water micro-alga is well known for its high protein content (70% of the dry matter). Among the algal proteins, it is worth noting the occurrence of phycobiliproteins in red and blue algae (blue phycocyanin in Spi rul i na, phycoerythrin in red algae) (Fan-jie et al., 1984). But the experiment of SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel) for algae is a formidable task because extraction of proteins from brown algae is difficult due to the occurrence of phenolic compounds (Ragan and Glombitza, 1986), Pigments and large amounts of polyanionic cell-wall mucilages mainly consisting of alginates (Mabeau and Kloareg, 1987). Phenolic compounds can destroy native protein structure as they attach and upon oxidizing conditions, couple covalently to them by tanning effect (Loomis and Battaile, 1966). Alginates from the cell wall form highly viscous solutions disturbing extraction and purification procedures for proteins. Furthermore they revealed, together with other components of brown algal cell walls, ion-exchange properties (Kloareg et al., 1987). Apart from nutritional study, Gel Electrophoresis of algal protein is also performed for purification and characterization of novel antibacterial protein from marine algae. Quite a distinct number of works was done on SDS PAGE of algae up to date. Two dimensional 1 Corresponding author: su_kalyanc@yahoo.co.uk (2) gels were run for whole cell extract of model alga Synechocystis sp. (Simon et al., 2002). Membrane protein separation of Chlamydomonas reinhardtii was done by Hippler et al., (2001). Red carotenoid astaxanthin was characterized in unicellular green algae Haematococcus pulvialis under environmental stress condition (Lorenz and Cysewski, 2000). In this study, we analyzed the protein content and protein profile of 8 species of marine macroalgae abundant in Sunderban mangrove estuary of India. MATERIAL AND METHODS Sample collection and preservation Eight species of benthic algae, which are found in large quantities throughout the ecosystem, were collected, in food grade plastic bags from Marichjhapi in the east, Canning, Gosaba and J harkhali in the central part, and Patharpratima and Dhanchi island in the western part of Sunderban (between latitude 2131-2240 North and longitude 8805-8906 East). The algal forms were hand picked from substratum like mud, concrete surface and bark and pneumatophores of trees. The samples were washed twice with seawater followed by fresh water and single distilled water to remove the adhering impurities, sand debris and epiphytes. Among these algal taxa four species (Rhi zocl oni um ri pari um, Enteromorpha intestinalis, Lola capillaris and Ulva lactuca) belonged to green algae, Dictyota ceylinica to brown algae and three species (Catenella repens, Polysiphonia mollis and Gelidiella acerosa) belonged to red algae. Extraction and estimation of protein The Lowry method (Lowry et al., 1951) was used for protein determination. The amount of protein present was determined comparing to authentic BSA standard (Sigma, USA) and expressed in terms of percentage of dry weight basis. Fig 1. Protein bands by SDS PAGE of the eight benthic macroalgae (M - Protein Molecular Marker, D - Dictyota ceylinica, G - Gelidiella acerosa, R - Rhi zocl oni um ri pari um, L - Lol a capi l l ari s, C - Catenel l a repens, P - Polysiphonia mollis, E - Enteromorpha intestinalis, U - Ulva lactuca) (3) Gel Electrophoresis of protein Electrophoresis of proteins in Poly acrylamide gel was carried out in buffer gels (non- denaturing) as well as in Sodium Dodecyl sulfate (SDS) containing (denaturing) gels. Seperation in buffer gels relied on both the charge and size of the protein whereas it depended only on the size in SDS gels. Poly acrylamide gels were formed by polymerizing acrylamide with a cross linking agent (bisacrylamide) in the presence of a catalyst (persulphate ion) and chain initiator (TEMED). Solutions were degased by evacuation prior to polymerization. The samples after mixing with equal volume of sample buffer were loaded in the wells and the gel was run starting with 60 V and then to 120 V. The plates were then disassembled and the gel was stained with silver stain. With respect to the marker, the molecular weights of the protein bands in the samples were estimated (Sambrook & Russell2001). RESULTS AND DISCUSSION The protein content showed marked individual variation (Table 1) with a highest average value of 40.87% of the dry matter in Lola and lowest, 3.33% of the dry matter in Dictyota. These values were consistent with earlier studies on other marine algae (Norziah and Ching, 2000). SDS PAGE revealed characteristic pattern of protein bands of all the eight benthic algae, with selected over expressed bands (Fig 1). Their results showed a high degree of homogeneity along with characteristic pattern of protein bands among all strains. This suggests that protein expression is a phenotypic character, which is regulated by both genotype and environmental factors and differences in protein profiles must be a reflection either of underlying differences in the regulation of gene expression or in post- translational modification of common proteins. Similar work was carried out by Rouxel et al., (2001) on four red algae, where he found six protein characteristic protein bands. Brown alga Dictyota showed the presence of total seven subunits, one between 97.4 kDa and 205 kDa, two between 43 kDa and 66 kDa, three prominent bands adjacent to band of ovalbumin at 43 kDa. Previously this protein was found by Rouxel et al., (2001) in C. crispus. Another band corresponding to carbonic anhydrase of 29 kDa was also observed. 29 kDa protein was earlier reported in Alexandrium catenella by Bustamante et al. (2003). In Gelediella, 4 bands were observed corresponding to ovalbumin at 43 kDa with trailings of protein bands at positions 205 kDa and between 97.4 kDa and 66 kDa. A glycoprotein noncovalently associated with cell-wall polysaccharide of the red microalga Porphyridium sp.of 66 kDa was reported by Prakash et al.(2004). Rhizoclonium, belonging to Chlorophyceae exhibited 6 bands with two bands corresponding to unknown molecular weight between 97.4 kDa and 205 kDa, one band between 97.4 kDa and 66 kDa two bands adjacent to protein carbonic anhydrase of 29 kDa. Another band of molecular weight below 3kDa was also observed in this alga. Lola again belonging to Chlorophyceae showed only 4 overexpressed protein bands out of which one corresponded to 43 kDa, two near 29 kDa and one below 3 kDa. Catenella, however showed a different pattern of protein bands. This might hint at its phylogenetic position also in Rhodophyceae. It showed a total of 6 bands, out of which, one was found higher than 205 kDa, one adjacent to 66kDa, one at 43 kDa, one between 20.1kDa and 29 kDa, one at 14.3 kDa and another at 6.5 kDa. Mainly 4 bands were found in Polysiphonia, a red alga. Two prominent bands were found with one between 205 kDa and 97.4 kDa and the other between 97.4 kDa and 66 kDa. Another band was seen between 20.1 kDa and 29 kDa. The most distinct band was found between 6.5 kDa and 3 kDa. Enteromorpha showed very interesting pattern of 4 clear bands. Three bands were between 43 and 66 kDa and one band of 14.3 kDa. Ulva however belonging to Chlorophyceae again showed 5 individual bands, one corresponding to Myosin band of the marker, one between 97.4 and 205 kDa, one bovine serum albumin band of 66 kDa, one of 29 kDa and one slightly more than 20.1 kDa. 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