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Date: Thursday 14th October 2010

Course Code & Title of Lab: BIOL 2361- Proteins and Amino Acids Aim: To determine the concentration of protein in a given standard protein solution via the Lowry protein assay and spectrophotometric analysis; To investigate the presence of protein in three unknown solutions via the usage of ninhydrin; To determine the presence of protein in a standard protein solution via the protein precipitation method using the solutions:10% Trichloroacetic acid solution, Saturated ammonium sulphate solution, 1M HCl, 1M NaOH, 2% Copper Sulphate solution (CuSO4), 5% Lead Acetate solution and Ice cold Ethanol; To separate and determine amount of different proteins in a solution by using gel filtration chromatography with sephadex-G-100 gel and to investigate the movement of glycine, glutamic acid and lysine solutions on filter paper using the electrophoresis method. Theory: Proteins are macromolecules formed by the linkage of amino acid groups via peptide linkages (Nelson and Cox 2008).

(Solomons and Fryle 2006)

The aim of protein purification is to isolate one particular protein from all the others in the starting material. Techniques utilized exploit the solubility, size, charge and specific binding affinity of the protein of interest (Hames and Hooper 2005). The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The principle lies in the reactivity of the peptide nitrogen with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteay phosphomolybdicphosphotungstic acid (yellow) to heteropolymolybdenum blue (blue) by the copper-catalyzed oxidation of aromatic acids/aromatic protein residues (Dunn 1992). Spectrophotometric analysis relies on the interaction of electromagnetic radiation with the matter of interest that is every compound has a distinct absorption spectrum which allows its identification. In addition to its identification, it is also possible to determine quantitatively the concentration of that compound (Dorey and Draves 1998). The relationship between absorbance and concentration is given by the Lambert-Beer Law : A= Lc
A is the absorbance is the molar absorptivity constant

L is the path length over which the light interacts with the sample in cm. c is the concentration

Precipitation is widely used for product recovery of biomolecules especially proteins. Precipitation is usually induced by addition of a salt or an organic solvent or by changing the pH to alter the nature of the solution. Protein solubility depends on several factors. It is observed that at low salt concentrations, solubility of the proteins usually increases slightly. This is termed Salting in. But at high concentrations of salt, the solubility of the proteins drops sharply. This is termed Salting out and the proteins precipitate out. Addition of an organic solvent results in a decrease in the dielectric constant of the medium. At low pH's, proteins have a net positive charge because the amide gains an extra proton. At high pH's, proteins have a net negative charge, due to the carboxyl on the protein backbone losing its proton. At their pI value, a protein has no net charge. This leads to reduced solubility because the protein is unable to interact with the medium and will then fall out of solution (Young 1994). In Gel filtration chromatography, molecules are separated on the basis of their size and shape. The protein sample is applied to a column of porous beads. Sephadex-G-100 is used in this experiment. Small molecules can enter the pores in the beads whereas larger or more elongated molecules cannot. Larger molecules move through the column first due to the less liquid accessible to them and are hence eluted first. The smaller molecules move slower through the column and elute later (Hames and Hooper 2005). When placed in an electric field, molecules with a net charge, such as proteins, will move towards one electrode or the other (Hames and Hooper 2005). Compounds bearing a net negative charge will move toward the anode whilst those bearing a net positive charge will move towards the cathode. Additionally, smaller molecules are deflected greater than larger molecules.

Fig. 1: Pictorial diagram of protein electrophoresis. The compound bearing the net positive charge moves towards the negative end and vice versa.

(Charles Sturt University, Chemistry Department 2002)

Procedure: 2

Procedure for the Lowry Assay: Reagents: 1. 2. 3. 4. 5. Standard Protein solution - 200g protein per mL i.e. 20mg per 100mL Lowry Reagent A 2% Na2CO3 in 0.1M NaOH Lowry Reagent B 0.5% CuSO4.5H2O in 1% sodium citrate Lowry Reagent C Folin-Ciocalteus Reagent

Twelve test tubes were obtained and labelled 1-12. 0, 0.1, 0.3, 0.5, 0.7 and 1mL of standard protein solution were pipetted into the test tubes twice. The volumes in the test tubes were made up to 1.0mL using water. 5.0mL of Lowry reagent C was added to all the tubes and left to stand for exactly 10minutes. 0.5mL of diluted Folin-Ciocalteus Reagent was then added and mixed. The tubes were left to stand for 30minutes at room temperature and the absorbance read at 750nm. A calibration curve of Absorbance reading at 750nm vs. g of protein was plotted. Ninhydrin Reaction: 4 drops of each of the 3 amino acids were placed on one filter paper and allowed to dry. One drop of ninhydrin solution was placed on each spot and gently warmed over a burner. The colours produced were noted. Procedure for protein precipitation: Reagents: 1. 2. 3. 4. 5. 6. 7. Standard protein solution 10mg/mL 10% Trichloroacetic acid solution Saturated ammonium sulphate solution 1M HCl 2% Copper Sulphate solution (CuSO4) 5% Lead Acetate solution Ice cold Ethanol

Seven test tubes were obtained and labeled 1-7. 3mL of standard albumin solution were added to each tube. 2mL of 10%TCA was added to tube 1; 2mL of saturated ammonium sulphate solution was added to tube 2; 2mL of 1M HCl was added to tube 3; 2 mL of 1M NaOH was added to tube 4; 1 drop of 2% CuSO4 was added to tube 5; 2 drops of 5% lead acetate was added to tube 6 and 10 mL of cold ethanol was added to tube 7.

Gel Filtration Chromatography: 3

0.2mL of blue dextran solution was added slowly to the column followed by 0.2mL of cytochrome c solution. The volume was allowed to penetrate the column until the fluid surface reached the top of the bed. 0.05M phosphate buffer was added to the column and filled to the top. The clamp was adjusted so that the flow rate was one drop every 4 seconds. The eluate was collected in a 10mL measuring cylinder. When the first band began to exit the tip of the column, the cylinder was removed and replaced with another 10mL measuring cylinder. Another measuring cylinder was used after the second band began to exit. The volumes of the first 2 cylinders were noted and the height of the column bed measured. Electrophoresis of Amino Acids: Reagents: 1. 2. 3. 4. NH4OH: Dilute 60mL of concentrated NH4OH to 600mL with 0.01M KCl. 10% Acetic Acid: Phosphate buffer A: Amino acids: 0.2% (w/v) solutions of glycine, glutamic acid and lysine preserved with a drop of toluene. 5. Ninhydrin solution 3 strips of filter paper were collected and a light pencil line was drawn across the center of each sheet. One end of the paper was marked X to be immersed into the anode compartment. 4 equally spaced circles were marked on the centre line for the amino acid samples, lysine, glutamic acid, glycine and a mixture of three. A capillary pipette was used to transfer a small amount of each sample into its appropriate spot. The paper was placed into the electrophoresis chamber and clips were used to secure the ends of the paper. The cover was placed on the chamber and buffer solution was allowed to creep up the paper until it was completely wet. The red terminal of the power supply was connected to the positive terminal of the chamber and the black terminal was connected to the negative terminal. The power supply was connected to an outlet and the switch turned on. The electrophoresis procedure was done for 40minutes and the current in milliamps noted. The paper was removed and dried. The strips were dipped into 0.3% ninhydrin solution and dried in an oven at 105C for 2 minutes. The electrophoresis procedure was repeated in the other 2 buffers.

The distance moved by each amino acid from the origin to the centre spot was measured and movement towards anode/cathode was recorded. The resulted were tabulated and graphed and the approximate isoelectric point of each amino acid determined.

References

Charles Sturt University, Chemistry Department. Charles Sturt University. 2002. http://www.hsc.csu.edu.au/chemistry/options/forensic/2965/ch993nov03.html (accessed October 10, 2010). Dorey, and Draves. "Spectrophotometric Determination of Total Protein-Biuret Method." University of Central Arkansas. May 1998. http://www1.gantep.edu.tr/~balci/protein%20%2816%29.pdf (accessed October 10, 2010). Dunn, M. J. 1992.Protein determination of total protein concentration. Oxford: IRL Press, Hames, David, and Nigel Hooper. 2005. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group, Nelson, David L, and Michael M Cox. 2008.Lehninger Principles of Biochemistry. United States of America: W.H. Freeman and Company, Solomons, T.W. Graham, and Craig B Fryle. 2006.Organic Chemistry. United States of America: John Wiley & Sons, Young, Derek R. Introduction to Biochemical Engineering . 1994. http://www.rpi.edu/dept/chemeng/Biotech-Environ/PRECIP/precpintro.html (accessed October 10, 2010).