1. Title: Lesson 5 B.7 Analysis of Proteins (SL and HL) Learning Objectives: – Describe methods to identify amino acid composition of an isolated protein – How to work out the pH of a buffer solution – Analysis of protein concentration

2. Analysis of proteins Who would find this useful? Clinicians – Analysis of protein levels can confirm pregnancy or a variety of diseases Food analysts – Analysis concentration and type of proteins in foods Pharmaceutical industry – Use altered protein levels to test efficacy of a drug Two main aspects of protein analysis: Amino acid composition of an isolated protein Protein concentration of a sample

3. Analysis of the amino acid composition of a protein Break peptide bonds in the protein structure through hydrolysis This is a reverse condensation reaction (common in digestion) Separating the resulting amino acid mixture can be done by: 1 Chromatography 2 Electrophoresis

4. 1 Chromatography Basic principle: Components have different affinities for two phases, a stationary phase and a mobile phase… The components are separated as the mobile phase moves through the stationary phase Amino acids are colourless in solution so are treated with a locating agent Paper chromatography (type of partition chromatography), which separates components on the basis of their different solubility in the two phases It is an example of qualitative analysis TASK: Write a step by step outline of (page 552 in the oxford textbook): How chromatography works How to identify different amino acids using Retention Factor (R f ) SL

5. 2 Electrophoresis Basic principle: Different charged particles will move different distances in an electric field Amino acids carry different charges depending on the pH, and these differences can be exploited to separate them when placed in a buffered solution at a particular pH… TASK: Use page 551 to: Explain why the amino acid solution needs to be placed in a buffered solution using your previous knowledge about amphoteric zwitterions… Write a step by step description of how the process works… Gel Electrophoresis MIT Video SL

7. Summary of effects of pH on charge This assumes that the R group is an uncharged group… Positively charged at low pH Negatively charged at high pH Intermediate pH where there is no net charge is called the isoelectric point  will not be able to move in an electric field With no net charge, there is minimum mutual repulsion between molecules so will be least soluble SL

8. HINT: Use the data booklet to check the isoelectric points of each amino acid. Refer to the previous slide to remind your self about charges and pH. SL

9. Determination of the pH of buffer solutions Buffers resist change in the pH of a solution on the addition of small amounts of acid or base (refer to chapter 8 to refresh on this…) The pH of a buffer solution, tat is its H + concentration, will depend on the interactions among its components. The equilibria that exist in the buffer will be: We can make two approximations: 1. Dissociation of the weak acid is so small that it can be considered negligible: 2. Salt is considered to be fully dissociated into its ions: Equilibrium expression for the acid is: therefore: (all concentrations must be equilibrium concentrations) HL

10. Substitute approximations: Which means: Take negative log of both sides: Basic buffer solutions, the equivalents equations are: These equations allow to know the pH of a buffer solution directly from: K a or K b value of component acid or base Ratio of initial concentrations of acids and salt used in buffer These equations (known as the Henderson- Hasselbalch equations) are given in section 1 of the IB Data booklet…

11. Urine analysis using a multiple test stick against a reference chart. Body fluids such as urine are effectively buffered for pH, so alterations in the urine pH can provide insight into medical conditions. From the example: When a buffer solution contains equal amounts in moles of acid or (base) and salts… becomes zero So pH=pK a (or pOH=pK b ) So to prepare a buffer of a specified pH, just choose an acid with a pK a value close to the required pH, and adjust concentrations if necessary. Just prepare by reacting the acid with enough strong alkali to get half into salt…

14. Analysis of the protein concentration of a sample Protein assays are investigative procedures used to measure the concentration of protein A common technique is UV-visible spectroscopy (UV-vis) Like other forms of spectroscopy, it depends on the fact that molecules interact with different parts of the electromagnetic spectrum according to their different chemical composition In this case… Visible and UV… Range of radiation is approx. 180-750 nm Enough energy to excite the electrons in the occupied higher energy levels in complex molecules When a full range of wavelengths of UV-vis radiation is passed through a sample, an absorption spectrum is obtained, which corresponds to an electronic transition… Absorbance spectrum of chlorophyll Peaks in the red and blue parts (high absorption) Very low absorption in the green light hence is green is transmitted giving the colour…

15. Absorption spectra are obtained from a spectrophotometer… A wavelength of maximum absorbance is selected for the analysis The amount of light absorbed (A, absorbance) at this wavelength is given by the relationship: Where: I 0 = the intensity of light before passing through the sample I = the intensity of light after pass through the sample The absorbance (A) can be linked to a law known as the Beer-Lambert law (which can be found in section 1 of the IB data booklet) A = log 10 (I 0 /I) RSC - UV-Vis Spectrophotometer in Action Video

16. Beer-Lambert Law Absorbance (A), depends on: The molar absorptivity, ε, defined as the absorbance of a 1.00 moldm -3 solution in a 1.00 cm cell at a specified wavelength Concentration of the solution, c Path length, l The Beer-Lambert law is expressed as: “ The absorbance of a compound is directly proportional to its concentration (at a fixed wavelength)” UV-vis Spectrophotometer Video

17. UV-vis procedure Involves reacting the sample with a reagent that generate a colour change which is dependent on the amount of protein present Promotes absorption in the UV-vis range Several different reagents can be used to react with the different side groups of the protein (e.g. peptide bonds, aromatic or basic groups etc…) A common reagent is biuret reagent, which generates a purple colour with reaction to peptide bonds

18. Converting absorbance data Use a calibration curve based on standard solutions with a known concentration of protein At least 5 dilutions of a standard known concentration are prepared They are treated with the appropriate reagent to generate colour Absorbance is measured at selected wavelength and plotted Absorbance of the sample solution is measured at same wavelength as standard solutions and protein concentration can be determined BSA = Bovine Serum Albumin (protein standard) Samples and standard solutions need to be treated in the same way w.r.t. type/amount reagent added, buffers, temp…

19. Full calibration curve The graph is linear at low concentrations, according to Beer-Lambert law

22. Solutions