Name: ID#:

Lab Partner: Date: Thursday 14th October 2010

Course Code & Title of Lab: BIOL 2361 Proteins and Amino Acids

Results: Experiment # 1 Lowry Test. Table 1: Absorbance readings at 750nm for varying protein concentrations Concentration of protein in solution Absorbance Average Absorbance (g) (750nm) (750nm) 20 0.045 0.044 20 0.043 60 0.157 0.173 60 0.188 100 0.243 0.259 100 0.274 140 0.336 0.353 140 0.369 200 0.444 0.449 200 0.453 Table 2: Absorbance readings at 750nm for unknown solutions containing an unknown concentration of protein Unknown Protein Absorbance (750nm) Average Absorbance (750nm) A 0.283 0.287 A 0.290 B 0.170 0.177 B 0.184 Experiment # 2 Ninhydrin Reaction Table 3: Colours produced upon addition of Ninhydrin to unknown Amino Acids Amino Acid Colour produced x yellow y purple z blue

Experiment # 3 Protein Precipitation Table 4: Appearance of Protein solution after the addition of various reagents Reagent Appearance of protein solution 2mL of 10% TCA On addition of TCA a white precipitate formed resulting in the colourless protein solution becoming cloudy. Upon heating the white precipitate became more concentrated at the top and below the tube leaving a colourless solution in between 2mL of saturated On addition of ammonium sulphate solution the colourless protein solution ammonium sulphate became translucent. Upon heating, a white precipitate formed which floated solution at the surface of a colourless solution below 2mL of 1M HCl On addition of HCl, the transparent, colourless protein solution became translucent. Upon heating, a white suspension formed that was slightly opaque 2mL of 1M NaOH On addition of NaOH, the colourless protein solution became translucent. Upon heating the translucent solution became colourless and transparent 1 drop of 2% CuSO4 On addition of copper sulphate a white precipitate formed in the colourless protein solution. Upon heating, the white precipitate was still present in the colourless solution 2 drops of 5% Lead On addition of lead acetate a white precipitate formed in the colourless Acetate protein solution. Upon heating the precipitate settled and became more concentrated at the surface and at the bottom leaving a colourless solution in between 10mL of cold Ethanol On addition of ethanol, a white precipitate was formed in the colourless protein solution.

Gel Filtration Chromatography Table 5: Volumes of eluates collected Colour of Eluate Volume Blue 3.5 Brown 1.5 Electrophoresis of Amino Acids Table 6: Distance moved by amino acids dipped in NH4OH Amino Acid Distance moved (cm) Lysine Glutamic Acid Glycine Mixture 3.1 6.8 5.8 3.0 and 6.9

The movement of amino acids was towards the anode Table 7: Distance moved by amino acids dipped in Phosphate Buffer Amino Acid Distance moved (cm) Lysine Glutamic Acid Glycine Mixture 1.9 0.7 0.6 0.6, 0.9 and 1.7

The movement of amino acids was towards the cathode Table 8: Distance moved by amino acids dipped in Acetic Acid Amino acid Distance moved (cm) Lysine Glutamic Acid Glycine Mixture 2.9 3.2 3.2 3.1

The movement of amino acids was towards the cathode Calculations: Protein concentration of the 2 unknowns: The Average Absorbance readings for A at 750nm was 0.287 Using the equation of the calibration curve: y = 0.0024x 0.287 = 0.0024x x = = 119.58

Therefore in 1mL of the unknown A, there were 119.58g of protein and in unknown B there were 73.75g of protein. Void Volume The void volume, Vo, is the volume of liquid between and outside the gel particles in the column. The void volume can be determined by measuring the elution volume of the macromolecule that is completely excluded. In this case, that macromolecule would be blue dextran since its M.wt is >1 x compared to13 000, the M.wt of cytochrome c. 3

The elution volume of blue dextran was 3.5mL. Therefore the void volume, Vo = 3.5mL. Bed Volume Volume of a cylinder = r2h Where h=15cm and r=0.5cm V= 15 x x V=11.78cm3
(Approximately 29.7%)

Discussion: 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). Using spectrophotometric analysis, the colour change of Folin-Ciocalteay Reagent can be measured and the protein concentration of a solution determined, see calculations. A calibration curve was plotted so that the concentration of the unknown samples can be determined by comparing the unknown to a set of standard samples of known concentration. Ninhydrin is used to detect ammonia or secondary amines; hence it can be used to detect the amino group on proteins. Upon reacting with amines, there is a colour change from colourless to deep blue (Nelson & Cox, 2008). In this experiment, amino acid X turned yellow, amino acid Y turned purple and amino acid Z turned blue. The amino acids proline and hydroxyproline turn yellow upon addition of ninhydrin therefore the unknown X could be either proline or hydroxyproline, the structures of which are shown in Fig. 1

(a) (b) Fig.1. (a): Structure of amino acid proline; (b) Structure of amino acid hydroxyproline (Nelson & Cox, 2008) The amino groups which react with the ninhydrin to produce the purple colour are involved in bonding with the R groups of these molecules and are hence unable to produce the purple colour, thus remaining yellow. 4

Protein precipitation allows for certain organic acids, solvents, heavy metal ions and concentrated neutral salts to react and cause the protein to precipitate out of the solution (Solomons & Fryle, 2006). TCA reduces the hydration layer around protein molecules via the dissociation of its chloride ion thus producing the acidic acetate ion. Competition between this chloride ion for hydrogen bonding between the protein and water in addition to the new pH causes precipitation. When the salt concentration of saturated ammonium sulphate is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result the protein-protein interactions are stronger than the solvent-solute interactions and the protein molecules coagulate by forming hydrophobic interactions with each other. On addition of HCl, a white precipitate was produced. HCl brings proteins to their pI point at which there is no charge. Therefore repulsive forces decrease and dispersive forces increase. Dispersive forces are responsible for the agglomeration of the protein molecules to form precipitate. Ethanol is a polar solvent and is hence capable of forming hydrogen bonds with water molecules. This reduces the number of water molecules interacting with the protein and hence results in stronger protein-protein bonds. This accounts for the white precipitate observed. In Gel filtration chromatography, molecules are separated on the basis of their size and shape (Hames & Hooper, 2005). In the experiment the molecules dealt with were blue dextran and cytochrome c. The blue dextran moved faster through the column than the cytochrome c. Blue dextran has a molecular weight of >1 x Da compared to13 000Da, the M.wt of cytochrome c. Therefore the blue dextran is a lot larger than the cytochrome c. In Gel Filtration Chromatography 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 hence the elution of the larger molecule, blue dextran, before the elution of the smaller molecule cytochrome c. The void volume of the column was calculated by determining the volume of elution of blue dextran, see calculations. Proteins can also be separated via electrophoresis. When placed in an electric field, molecules with a net charge, such as proteins, will move towards one electrode or the other. 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. (Hames and Hooper 2005). The amino acids investigated were Glutamic acid M. Wt of 147.13, Lysine M. Wt of 146.19 and Glycine M. Wt of 75.07. When placed in the alkali ammonium hydroxide solution all the amino acids moved towards the anode, however, glutamic acid (see Fig.1) moved the furthest distance of 6.8cm. 5

The distance moved by glutamic acid can be explained by its net ionic charge. When placed in the alkali solution it would have gained a net charge of -2 via the loss of hydrogens from the COOH groups thereby assisting in the rapid movement in the electric field.
Fig.1: Structure of Glutamic Acid (Nelson & Cox, 2008)

Glycine moved the 2nd fastest with a distance of 5.8cm. This was due to its low molecular weight hence its rapid movement in the electric field. Lysine did not move as far since it was almost the same molecular weight as glutamic acid, however it only contained a -1 charge in the alkali solution. In the acidic acetate buffer all the amino acids moved towards the cathode, however, lysine (see Fig.2) moved the greatest distance due to its net ionic charge of +2. In acidic environments hydrogen atoms are gained by the NH2 groups on the lysine molecule thereby resulting in a net +2 charge. Fig.2: Structure of a lysine molecule

In the phosphate buffer all the amino acids moved towards the cathode. Glycine moved the slowest due to its neutral charge in the solution however, lysine moved the greatest distance due to it being slightly lighter than the glutamic acid molecule. In the buffer solution the charges of the molecule would have no effect due to the system being buffered.

Additional Discussion: 1. Gel filtration chromatography separates proteins according to size. Large proteins emerge from the column sooner than small ones. The solid phase consists of cross-linked polymer beads with pores of a particular size. Large proteins cannot enter the pores and so take a short path through the column, around the beads. Small proteins enter the pores and slowly move through the column (Nelson and Cox 2008). Since gel filtration chromatography separates proteins on the basis of size, it can be used to separate small protein molecules from larger protein molecules. The larger protein molecules would be eluted first from the column whereas the smaller molecules would take a longer time. Another method used to separate small molecules from proteins would be dialysis. In dialysis, the partially purified extract is places in a bag made of a semi permeable membrane. When this is suspended in a large volume of buffered solution of appropriate ionic strength, the membrane allows the exchange of salt and buffer but not proteins. Dialysis retains large proteins within the membranous bag while allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium with the solution outside the membrane (Nelson & Cox, 2008). 6

2. Small peptide fragments can be separated via ion exchange chromatography and affinity chromatography. Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge (Nelson & Cox, 2008). It involves the use of a column packed with specific bead types with which proteins will bind. The proteins that have been bound to the beads on the column can then be washed away using a counter ion solution. Affinity chromatography makes use of the specific, high affinity, non-covalent binding of a protein to another molecule (Nelson & Cox, 2008). This also makes use of a column; however the contents are only able to bind specific proteins. 3. Electro-osmosis is the motion of polar liquid through a membrane or porous structure under the influence of an applied electric field. In vascular plant biology, electro-osmosis is used to explain the movement of polar liquids via the phloem. 4. Electrophoresis is the process by which proteins are separated in an electric field on the basis of their net charge and size (Hames & Hooper, 2005). Factors affecting electrophoresis are: Concentration of proteins within solution: The greater the concentration of a particular protein, the more likely it is to be detected. Some classes of analyte cannot be separated by this effect because they are neutral or are at their isoelectric point in certain solvents. Some analytes may not differ significantly in their electrophoretic mobility and hence cannot be distinguished. Buffer/ solvent concentration: High concentrations of solvent reduce migration speed and enable the separation of smaller proteins. Voltage: The higher the voltage, the faster the proteins will move thereby disabling the detection of small proteins. However, larger proteins will be detected faster in high voltages. 5. Glycine:

Glutamic Acid:

Lysine:

6. The greater the net charge, the greater the mobility. E.g. see Results: Table 6: Distance moved by amino acids dipped in NH4OH. Glutamic acid moved the greatest distance of 6.8cm due to its net charge of -2 in the alkali solution. Lysine and Glycine had a net charge of -1. Due to the glutamic acid being a charged species, the ease of movement throughout the electric field will increase therefore accounting for its rapid movement and great distance moved.

References

Dunn, M. J. 1992. Protein determination of total protein concentration. Oxford: IRL Press. Hames, D., & Hooper, N. 2005. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group. Nelson, D. L., & Cox, M. M. 2008. Lehninger Principles of Biochemistry. New York: W.H. Freeman and Company. Solomons, T. G., & Fryle, C. B. 2006. Organic Chemistry. United States of America: John Wiley & Sons.