E002 Corrected MODULE 2 PR PDF

Module no. 2 Isolation of Proteins and Determination of Concentration by Spectrophotometry METHOD 1: Isolation of crude ovalbumin from egg white by ammonium sulfate precipitation Salting out o...

Module no. 2 Isolation of Proteins and Determination of Concentration by Spectrophotometry METHOD 1: Isolation of crude ovalbumin from egg white by ammonium sulfate precipitation Salting out o Process of precipitating proteins by saturating solution with ammonium sulfate o Ammonium sulfate dissociates into NH4+ and SO42- ▪ Attracts water molecules through ion-dipole interactions ▪ Proteins will be left out of solution since very few water molecules available for association Ions disrupt H-bonding between water and proteins Ion-dipole > H-bonding ▪ Proteins will aggregate then precipitate out of the solution o Presence of salt increases the ionic strength (μ) of the solution ▪ Measure of the concentration of ions in the solution ▪ Results from the fact that ions have an electric charge that can attract or repel each other This attraction and repulsion result in particular behavioral ways of ions o Increase in ionic strength decreases the solubility of proteins o ↑ [salt], ↑ μ, ↓ protein (and other hydrophobic molecules) solubility ▪ Causes precipitation Ammonium sulfate Salt with diffused charge NH4+ diffuses the positive charge such that only about a quarter of a positive charge is on any singe hydrogen SO42- diffuses the negative charge such that each oxygen has approximately one half of a charge Diffusion of charge prevents maximal disruption of the protein or polypeptide backbone structure Ion-ion attractive forces are not enough to disrupt protein folding (except in unstable polypeptides) o The ammonium and the sulfate gently strip away water molecules Proteins with fewer number of charged groups are easier to precipitate Solid ammonium sulfate when lower amount is required Liquid ammonium sulfate is used to prevent premature denaturation Advantages o Available in pure form o High solubility in water o Cheap o Has high ionic strength ▪ Two moles of ammonia and one mole of sulfate o Not strongly oxidizing or reducing Do not use lithium chloride or salts with heavy metals o Li+ and Cl- are densely charged ions with small ionic sizes o Ions strongly attract charged side chains of amino acids, which disrupts the polypeptide backbone since amino acids will instead be attracted to the ions instead of each other Egg white 88.5% water, 10% proteins o 54% ovalbumin, 12% ovotransferrin, 3.5% ovomucin, 3.4% lysozyme Highlights of the procedure Use of cheesecloth o Filters out emulsions that may be incorporated in the filtrate (increases impurity) o Prevents contamination that results when normal filter paper is used Addition of acetic acid o Precipitates most probably ovomucin (IpH – 4.75), which is responsible for the gel-like properties of egg white Gradual addition of ammonium sulfate with continuous stirring o Ensures that the whole solution is saturated o Prevents premature precipitation of ovalbumin Use of powdered ammonium sulfate o Increases surface area which facilitates faster precipitation o More contact with water 0-40% saturation o Precipitates out other unwanted proteins (most probably globulins) ▪ Insoluble in dilute salt solutions Use of ice bath o Prevents possible denaturation of proteins o Facilitates precipitation of ovalbumin due to the lowered temperature 40-60% saturation o Precipitates ovalbumin o Other proteins might still be included, but the majority is ovalbumin METHOD 2: Isoelectric precipitation of casein Isolation of proteins based on their isoelectric point At IpH, there is no net charge o No repulsion present between protein molecules o Formation of agglomerate or precipitate is encouraged o Point of lowest protein solubility and highest amount of precipitate formed At pH less than or greater than the IpH, there is a net charge o Repulsion is present Page 1 of 4  o There is little to no precipitation Highlights of the procedure Determining IpH o Observe test tube with the highest turbidity (indication of decreased solubility) o Theoretical IpH is 4.7 Different buffer systems used o To create variation in pH and better observe the relationship between solubility and pH Other methods of determining IpH Isoelectric focusing (IEF) o An electrophoretic separation method o Movement of amphoteric molecules (have both acidic and basic properties) based on their charge as defined by the pK a values of proton-accepting sites within a molecule o Separates proteins according to IpH Chromatofocusing o Allows resolution of single proteins and other ampholytes from a complex mixture according to differences in IpH o Medium used is a resin with charged molecules Determination of protein concentration by spectrophotometry Spectrophotometry o Visible light range: 380 nm (violet) to 750 nm (red) Transmittance (T) o Fraction of incident radiation transmitted after passing through the solution o Usually expressed in percentage (ratio of light that reached the detector after passing through the sample and the amount of light coming from the radiation source before being absorbed) Absorbance (A) o Inverse of transmittance o Measurement of light absorbed by a substance o Absorbance and transmittance have a logarithmic relationship ▪ ↓ T, ↑ A, ↑ concentration Bradford assay A direct, dye-binding method of determining protein concentration Requires an acidic solution of Coomassie Brilliant Blue G-250 o If basic or neutral, the maximum absorption will be different, and the dye will not bind to the protein o If basic, the dye is green and has a maximum absorption of 650 nm Under strongly acidic conditions, the dye is most stable as a doubly protonated red (or pale orange red) form o Not yet bound to proteins o Maximum absorption is 470 nm o Acidity is provided by 85% (w/v) phosphoric acid o Ethanol is used as solvent Upon binding, the dye is stable as an unprotonated, blue form o Maximum absorption is 595 nm Coomassie Brilliant Blue G-250 binds to positively charged (basic) amino acid residues such as arginine, histidine, phenylalanine, tryptophan, and tyrosine at acidic pH There are probably weaker interactions between the dye molecules and hydrophobic, aromatic amino acid residues Proteins having more than an average number of interacting residues show higher sensitivity and higher absorbance at 595 nm Exact mechanism of binding is still not yet fully understood After the addition of the dye, a red shift will occur o Maximum absorption is changed from 465 nm to 595 nm (increase in terms of wavelength) o The development of the blue color will finish in around two minutes o This shift is used to colorimetrically determine protein content Advantages o Quick and easy to perform o Inexpensive o Extremely sensitive and produces fairly accurate results ▪ Can detect proteins in the range of 1 to 20 μg o Reproducible o Compatible with most buffers, solvents, salts, reducing agents, and chelating agents in the solution o Very few interferences by non-proteins components ▪ Can increase or decrease absorbance ▪ Interferences are Triton X-100, sodium dodecyl sulfate, commercial glassware detergents Disadvantages o Color is absorbed by glassware, cuvettes, clothing, and skin o Incompatible with surfactants (causes precipitation) o Coomassie is highly acidic and cannot be used for proteins with poor acid solubility o High sensitivity to basic and aromatic amino acids Parts of a spectrophotometer Light source → monochromator → sample holder → detector → read-out system Beer-Lambert’s Law There is a directly proportional relationship between the light absorbed and the number of molecules (concentration of substance in solution) There is a linear relationship between absorbance and concentration o Allows for easy determination of concentration of a sample from a standard curve o A = abc ▪ A is absorbance Page 2 of 4  ▪ a is molar absorptivity or molar extinction coefficient (L/mol-cm) ▪ b is path length (cm) ▪ c is sample concentration (mol/L) Limitations (causes of nonlinearity) o At very high concentrations, the graph of plateaus o Can only be used at dilute concentrations ▪ At high concentrations (> 0.01 M) due to electrostatic interactions between molecules in close proximity o Can only be used for monochromatic light (one wavelength) ▪ Nonlinearity due to non-monochromatic radiation is minimized by using a relatively flat part of the absorption spectrum ▪ Only λmax is used ▪ Otherwise, the absorbance is split (will result in lower or higher absorbances) o Light that reaches the detector should only be transmitted light (no stray light) o Only one absorbing species should be present (avoids positive error) o Changes in refractive index at high analyte concentrations Standard curve Linear graph produced from the plot of absorbance versus concentration as governed by Beer-Lambert’s law Constructed form the absorbance of several standard solutions that have known identities and concentrations o Usually BSA or bovine serum albumin ▪ Globular protein used to determine estimated concentration as a standard ▪ Derived from cow blood and undergoes three different purification methods (high purity) ▪ Very useful in different biochemical applications because it is stable ▪ Small and can be easily stored ▪ Stable at room temperature ▪ Highly pure sample and accessible at low cost Determination of protein content Purpose o Used to test activity of protein o Reports progress of purification ▪ Purification can decrease the concentration of proteins in the sample ▪ Determining the protein content is a marker of when to stop further purification o Identifies significant amount of contaminating proteins in sample Errors in the experiment Instrumental and personal errors Impurities Absorbing interferences Other methods of determining protein concentration How to choose the appropriate assay o Amount and concentration of protein ▪ Different assays have different sensitivities o Specificity of the assay o Interfering chemicals o Ease and reliability Warburg-Christian method o Relies on relative absorbance of proteins at 280 and 260 nm ▪ Absorption of tyrosine and tryptophan at 280 nm ▪ Absorbance at 260 nm is determined as correction for the absorbance of nucleic acids o Advantages ▪ Fast (requires only a solution of protein, no dyes or complexation) ▪ Non-destructive to sample o Disadvantages ▪ Nucleic acids absorb at 260 nm and can interfere with absorbance ▪ Presence of UV-absorbing compounds ▪ May be inaccurate if the protein has an unusual amino acid composition since the method depends on only two amino acids Biuret reaction o General test for peptide chains of at least three amino acids (i.e., 2 peptide bonds) o Under alkaline conditions, proteins with two or more peptide bonds form a purple complex with copper salts in reagent o Components of reduction ▪ 1.5 g CuSO4 5H2O (source of Cu2+, which interacts with nitrogen involved in peptide bonds) ▪ 6.0 g sodium potassium tartarate (prevents reduction of Cu 2+ to Cu since Cu2+ has the ability to interact with the two lone pairs in nitrogen) ▪ 500 mL distilled water (diluent) ▪ 300 mL NaOH (strong base that maintains alkaline conditions and enhances the complexation of Cu 2+ with peptide nitrogen since it is stable in basic conditions) o ↑ color intensity, ↑ # peptide bonds, ↑ [protein] o Limitations ▪ Low sensitivity and accuracy ▪ High presence of interferences Buffers that are peptide in nature Reducing sugars react with Cu2+ to produce a brick red precipitate NH3 forms complexes with Cu2+ Page 3 of 4  Lowry method o Based on the Biuret reaction with additional steps to increase sensitivity o Reagent ▪ Biuret + Folin-Ciocalteau Formation of Biuret complex Folin-Ciocalteau reagent o Phosphotungstic and phosphomolybdic acid (inorganic compounds) o Further stabilizes Cu2+, oxidizes Cu to Cu2+ o A yellow reagent that interacts with side chains of Tyr, Trp, and Cys to produce a blue- green color due to the formation of heteropolymolybdenum blue that can be detected between 650 to 750 nm o Advantages ▪ Highly sensitive (100 times greater than that of Biuret assay) ▪ Gives accurate results o Disadvantages ▪ More common substances can interfere with the method ▪ Folin reagent is reactive for only a short period of time after addition ▪ Method is complicated and requires more steps and reagents than other assays ▪ Destructive to proteins (proteins cannot be recovered for other assays) ▪ More time is required Page 4 of 4