Exercise 2 Fish Muscle Protein Introduction The protein content of fish muscle varies with species, season and

the environments. High levels of protein are found in the feeding around the spawning season. There are obvious incentives in using fish with high protein content. The protein in the sarcoplasmic fraction is excellently suited to distinguish fish species, as each species has a characteristic band run when separated by isoelectric focusing method or electrophoresis. For example, one might be able to use this technique to detect the substitution of inexpensive fish for an expensive fish. In this experiment, sarcoplasmic muscle proteins are extracted with a 0.15 M salt solution, the protein content of the extract is measured by the BCA assay. In which the protein present reduces cupric ions to cuprous ions under alkaline conditions. The cuprous ions react with the BCA reagent to give a purple colour that can be quantified spectrophotometrically and related to the protein content. (Suzanne, 2010) Objective To extract the protein from the muscles of patin fish (freshwater) and siakap fish (saltwater) and measure the protein content of the extracts. Materials SAMPLE EXTRACTION Sodium chloride, NaCl Sodium phosphate, monobasic (NaH2PO4.H2O) Blender 1 x 200 ml beaker 1 x 15 ml centrifuges tubes 1 x funnel 1 x test tube Filter paper Patin and Siakap Fish PROTEIN DETERMINATION (BCA Assay Kit) Kit Contents : BCA Reagent A, 500 ml BCA Reagent B, 25 ml Albumin Standard Ampules, 2 mg/ml, 10 x 1 ml Centrifuges tubes Micropipette tips 37 oC waterbath Cuvettes Spectrophotometer

Method Part 1 : Preparation of Reagent and Sample Processing Sample Extraction Buffer 1. 500 ml of extraction buffer was prepared which contain 0.15 M sodium chloride and 0.05 M sodium phosphate at pH 7.0 Sample Preparation 1. 40g of fish muscle were blended in 120ml of extraction buffer. 2. 10 ml of the muscle homogenate was poured into 15 ml centrifuge tube. 3. The sample is centrifuged at 2000 x g for 15 minutes at room temperature. 4. The supernatant is filtered using Whatman No.1 filter paper and collected in a test tube and capped. 5. The filtrate is stored at 4 oC for 1 week. Part 2 : Analysis BCA Protein Assay 1. The Working Reagent for the BCA assay was prepared by combining Pierce Reagent A with Pierce Reagent B, 50:1 (v/v), A:B. 10 ml of Reagent A and 0.2 ml Reagent B were used to prepare 10.2 ml of Working Reagent. 2. Dilution of 1:10 and 1:20 of supernatant was prepared in extraction buffer in a final volume of 1ml Dilution 1: 10 1: 20 Fish extract ml 100 l 50 l Extraction Buffer 0.9 ml 900 l 950 l

3. Duplicates were prepared for each reaction mixture of diluted extracts and BSA standards as indicated in the table below. Tube Identity Blank Std 1 Std 2 Std 3 Std 4 Std 5 Sample 1:10 Sample 1:20 ddH2O 50 45 40 20 10 0 25 25 BSA std (l) 0 5 10 30 40 50 Fish extract (l) 25 25 Working reagent (ml) 0 0.142 0.261 0.990 1.659 0.907 0.485 0.129

4. Each reaction mixture was mixed with a vortex mixer, and then incubated in a water bath at 37C for 30 min. 5. The absorbance of each tube at 560nm were obtained and recorded.

6. A standard curve was plotted, absorbance at y-axis and protein concentration on x-axis. Equation of the line for the standard curve was determined. 7. The protein concentrations were calculated based on the equation of the line. Results PATIN FISH Tube identity Blank S1 S2 S3 S4 S5 Sample 1:10 Sample 1:20 ddH2O (l) 50 45 40 20 10 0 25 25 BSA Std (l) 0 5 10 30 40 50 BSA concentration (mg/ml) 0 0.2 0.4 1.2 1.6 2.0 25 25 Absorbance (O.D) 0 0.142 0.261 0.990 1.659 0.907 0.485 0.129

SIAKAP FISH Concentration of fish protein: 1:10 = 5.546 mg/mL 1:20 = 4.684 mg/mL

Graph
1.8 1.6 1.4 BSA Concentration (mg/ml) 1.2 1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Absorbance (O.D)

Graph of BSA Concentration against Absorbance (560nm) Equation of line y = 0.9704x + 0.0876 Calculation Concentration of a) Sample 1:10 Absorbance value, x = 0.485 x 10 Concentration = 0.9704(4.85) + 0.0876 = 5.582 mg/ml

b) Sample 1:20 Absorbance value, x = 0.129 x 20 = 2.58 Concentration = 0.9704(2.58) + 0.0876 = 4.256 mg/ml

Discussion a) In BCA analysis, Proteins reduce alkaline Cu (II) to Cu (I) in a concentration dependent manner. Bicinchoninic Acid (BCA) is a highly specific chromagenic reagent for Cu (I) forming a complex with an absorbance maximum at 560 nm. Because of this property, the resultant absorbance at 560 nm is directly proportional to the protein concentration (Aldrich, 2013) b) In BCA analysis, two main steps are involved. First is the biuret reaction, where blue colour formed which is due to the reduction of Cu2+ to Cu+. Second is the chelation of BCA with the cuprous ion (Cu+),where purple color is formed. The purple coloured reaction product is formed by the chelation of two molecules of BCA with one cuprous ion (Thermo Scientific, 2013). c) The concentration of fish muscle against the absorbance value at 560nm shows a proportional increase. This graph is plotted with the values of absorbance 5 standard solution and blank as a control. By plotting the graph, linear regression line can be obtained which is used to calculate the sample fish concentration. From the calculation, we have 5.582 mg/ml for 1: 10 dilution and 4.256 mg/ml. During the calculation, the x value is multiplied by 10 and 20 which are the dilution factors. d) Two dilution of fish extract was prepared so that we could get an absorbance reading that is within a range of the protein concentration e) From our analysis, the protein concentration of patin fish is higher in 1:10 dilution and lower in 1:20 dilution compared to siakap fish. Each fish have different protein concentration and in this case, the protein concentration of 2 fish sample differ may due to season and the environmental condition. There might be different in the protein concentration of saltwater fish and freshwater fish. In our analysis, the patin concentration is different in 1:10 dilution and 1:20 dilution compared to siakap may due to error during carrying out the experiment. f) Another method that can be used for determination of protein content in food sample is Lowry method. Lowry reaction consists of the Biuret reaction followed by the reduction under alkaline condition using a reagent. Copper ions facilitate the reduction process.(Folin, 1927) The difference between BCA and Lowry is that in Lowry, the method relies on the color development from the Biuret reaction and from the reduction of an arsenomolybdate reagent (the FolinCiocalteau reagent) by the tyrosine and tryptophan residue in the treated protein while in BCA bichinchoninic acid is used. The colour in Lowry method is blue compared to BCA which is purple and the absorbtion in Lowry method is around 750nm compared to BCA which is 560nm (Krohn, 2002)

Conclusion The protein concentration of patin fish and siakap fish were obtained. In sample 1:10, the concentration of patin fish is higher compared to siakap fish, while in sample 1:20, the concentration of siakap fish is higher than patin fish.

References Suzanne S.N. ,2010 Analysis Laboratory Manual, Food Science Texts Series, pp 315;The Ohio State University : USA Sigma-Aldrich, 2013 Protein Determination by the Bicinchoninic Acid (BCA) Method [Online] Available : http://www.sigmaaldrich.com/life-science/metabolomics/enzymeexplorer/protein-determination-by-the-bicinchoninic-acid-bca-method.html Thermo Scientific, 2013 Chemistry of Protein Assays [Online] Available : http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0CB764EAB3A339 Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc Folin, O. and Ciocalteau, P. (1927) Journal of Biology and Chem. 73:627