Experiment VII: Determination of Protein Contents by Spectrophotometry

EXPERIMENT VII DETERMINATION OF PROTEIN CONTENTS BY SPECTROPHOTOMETRY Basic theory Determination of protein levels is a routine process used in Biochemistry laboratories. There are several common methods Determination of protein levels, both using titration spectrophotometric methods, each of which has its advantages and disadvantages. The best and most appropriate method for a measurement depends on several factors such as the amount of sample available, the time available for measurement, the size of the protein content in the sample, the available spectrophotometer (Visible or UV). Election Spectrophotometrically, protein levels can be determined using the Biuret method and Lowry method which will be carried out in this practicum. Determination of protein in food ingredients or animal tissue is often carried out by determining the total nitrogen content in the sample (Kjeldahl method N determination). In the method we assume that all the nitrogen determined comes from protein. Although this is not always the case (there are non-protein components that also contain N, for example nucleotides, urea and others), in practice the Kjeldahl method is often used because for certain purposes the results are quite satisfactory. This method for determining total N levels consists of three stages, namely; The Destruction Stage is to liberate all the nitrogen in the protein to become ammonium sulfate, the Distillation Stage is to break down the ammonium sulfate into ammonia so that it can be separated by distillation, and the last is the Titration Stage to determine the ammonia content. Biuret method Biuret determination of protein content is based on measuring light absorption by the purple complex compound formed between Cu2+with the C=O and NH groups of the peptide chain in basic conditions. When determining the protein concentration in a sample spectrophotometrically, measurements must also be made of several standard protein solutions that have a certain concentration range where the concentration of the protein sample is within that range. Protein is first added to the test tube. All tubes must have the same final volume and stir or mix well after each addition of the substance/reagent. Color producing reagents are always added last and Usually a certain time interval is required for a complete color reaction to occur. Reactor : - Biuret's reagent :Dissolve 1.5 g CuSO4.5H2O and 6.0 g sodium potassium tartrate (NaKC4O6.4H2O) into approximately 500 mL of water in a 1 liter volumetric flask. Then add 300 mL of 10% NaOH while shaking. And finally add distilled water until the limit mark. This blue solution can be stored for a long time. If the manufacture is not good, black deposits or black deposits can form red (cannot be used because it will interfere with absorption). - Protein standard solution :Make a solution of pure serum albumin or casein in water with a concentration of 1 - 10 mg per mL. To facilitate solubility add a few drops of 3% NaOH. Procedure: Pipette 1 mL of protein solution containing 1 to 10 mg per mL of protein into a test tube. Add 4 mL of Biuret reagent. Shake and let sit for 30 minutes at room temperature. Read the absorption at 540 nm. Assignments and Questions: 1. Construct a standard curve and determine the protein content of the given sample. 2. What compounds can interfere with this Biuret determination method? Lowry Method Phenolic groups detection reagent used in determining protein concentration by Lowry (1951) which became known as the Lowry method. In its simplest form, the Folin-Ciocalteu reagent can detect tyrosine residues (in proteins) because the phenolic content in these residues is able to reduce phosphotungstate and phosphomolybdate, which are the main constituents of the Folin-Ciocalteu reagent, to tungsten and molybdenum which are blue. The results of this reduction show a wide absorption peak in the red region of the visible light spectrum (600-800 nm). such as the Folin-Ciocalteu reagent The sensitivity of the Folin-Ciocalteu method experienced a significant improvement when combined with Cu ions (Biuret method). The Cuprotein complex produced by the Biuret reagent will cause reduction of phosphotungstate and phosphomolybdate in the Folin-Ciocalteu reagent. Approximately 75% of The reduction process that occurs is caused by the presence of the Cu-protein complex, while tyrosine and tryptophan residues reduce the remaining 25%. Solutions containing 20 to 200 -g of protein can be determined by this colorimetric method. Folin-Ciocalteu reagent is a complex composition obtained by reflux heating Na-tungstate and Na-molybdate with ortho phosphoric acid. Apart from that, other components are also included to increase the stability of the reagent, which under normal conditions is pale yellow in color. Reactor : Reagent A :2% Na2CO3in 0.1 N NaOH. Reagent B :0.5% CuSO4.5 H2O in 1% Na or K tartrate. Reagent C :Mix 50 mL of reagent A with 1 mL of reagent B. Discard after 1 day. Reagent D :Dilute Folin-Ciocalteu reagent with distilled water to 1 N in acid. Folin-Ciocalteu reagent :Reflux for 10 hours the following mixture: - 100 g sodium tungstate or sodium wolframate - 25 g sodium molybdate - 700 mL distilled water - 50 mL 85% phosphoric acid and 100 mL concentrated HCl Add:150 g lithium sulfate - 50 mL distilled water - a few drops of bromine water minutes to remove Bring to a boil 15 minutes to remove excess bromine (do not use condenser) . Dilute to 1 liter and filter. The solution must not be greenish in color. Determine the acid concentration by titrating 1N NaOH with phenolphthalein indicator. Protein Standard Solution:Make a standard solution of BSA protein ( Bovine Serum Albumine ) which contains 20 – 200 -g protein. Procedure: 1. Make a mixture of a standard protein solution with water until the volume is reduced not exceed 1.0 mL. Also mix the protein sample to be measured with water so that the final volume is 1.0 mL. (see Table III.1). 2. Add 5 mL of reagent C (Biuret Reagent) into each tube. 3. Incubationappropriately 10 minutes at room temperature. This time is too long critical. Use a stop watch (turn on the start) when adding the Biuret solution on tube 1, wait until a certain time interval (at least 30 seconds) before Add Biuret solution to tube 2, and so on. After 10 minutes, add 0.5 mL of reagent D (Folin– Ciocalteu reagent) to each tube. Shake immediately with a vortex or stirrer. 4. Incubate for 30 minutes at room temperature. This incubation time can begin (start) after adding/mixing reagent D into the last tube. 5. Read the absorbance at - = 700 nm with a spectrophometer using tube 1 as a blank. Question : 1. Make a standard curve and determine the protein concentration of the sample solution. 2. What are the advantages and disadvantages of Lowry's method compared to other methods of determining protein levels?