1. PROTEIN ANALYSIS

2. Introduction Proteins are polymers of amino acids Major structural components of many natural foods Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of proteins in foods.

3. Determination of Protein Concentration 1. Kjeldahl Method A food digested with strong acid so that it releases nitrogen which can be determined by a suitable titration techniques Does not measure the protein content directly Conversion factor is needed to convert the measured nitrogen concentration to a protein concentration

4. Principles A.Digestion Sample is heated in the presence of sulfuric acid (oxidizing agent), anhydrous sodium sulfate, and a catalyst. Digestion converts any nitrogen in the food into ammonia and other organic matter to CO2 and H2O Ammonia gas remains in acid solution because it is in the form of ammonium ion which binds to the sulfate ion to form ammonium sulfate

5. B. Neutralization The solution in the digestion flask is then made alkaline by addition of sodium hydroxide which converts the ammonium sulfate into ammonia gas The ammonia gas moves out into receiving flask which contains an excess of boric acid In receiving flask, ammonia gas converts to ammonium ion and boric acid to borate ion.

6. C. Titration Titrate with standard sulfuric or hydrochloric acid using a suitable indicator The concentration of hydrogen ions required to reach the end-point is equivalent to the concentration of nitrogen Once nitrogen content has been determined, it is converted to a protein content using appropriate conversion factor.

7. Advantages: universal high precision good reproducibility Disadvantages: does not give a measure of true protein different proteins need different correction factors the use of concentrated sulfuric acid at high temperature time consuming

8. 2. Enhanced Dumas Method Principles: a sample of known mass is combusted in a high temperature to release CO2, H2O and N2 Nitrogen content is measured by separating N2 from CO2 and H2O using a column Also used conversion factor to determine protein content

9. Advantages Faster Doesn’t need toxic chemicals or catalysts Easy to use Samples can be measured automatically Disadvantages High initial cost Does not give a measure of true protein Small sample size make its difficult to obtain a representative sample

10. 3. Methods using UV-Visible Spectroscopy Use either the natural ability of proteins to absorb (or scatter) light or chemically or physically modify proteins to make them absorb (or scatter) light in the region. Calibration curve of absorbance versus protein concentration must be built first Main difference: the chemical groups which are responsible for the absorption or scattering of radiation

11. Direct Measurement at 280 nm Principle: tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm Use the same w/length to measure protein concentration Advantages: simple to carry out, non-destructive and no special reagents are required Disadvantages: nucleic acid absorb strongly at 280nm To overcome: measure the absorbance at two different w/lengths

12. Biuret Method A violet-purplish color is produced when cupric ions (Cu 2+ ) interact with peptide bonds under alkaline conditions Absorbance is read at 540 nm Advantages: no interference from materials that absorb at lower w/lengths and less sensitive to protein type Disadvantages: low sensitivity

13. Lowry Method Combines biuret reagent and Folin-Ciocalteau phenol reagent which reacts with tyrosine and tryptophan residues in proteins Gives a bluish color which can be read at w/length between 500 – 750 nm More sensitive to low concentrations of protein than biuret method

14. Dye Binding Methods A known excess of a negatively charged dye is added to a protein solution which are positively charged (by adjusting the pH) Bound dye and protein form an insoluble complex while the unbound dye remains soluble The amount of protein present proportional to the amount of dye that bound to it.

15. Turbimetric Method Proteins from precipitate by addition of certain chemical e.g. trichloroacetic acid The concentration of protein is determined by measuring the degree of turbidity

16. Advantages: Rapid Simple to carry out Sensitive to low protein concentration Disadvantages: Time consuming and laborious Absorbance depends on the type of protein analyzed difficult to quantitatively extract proteins from certain types of food e.g. processed food

17. Other instrumental techniques Bulk physical properties Density – protein has the greatest density. So increase in protein content will increase density of food Refractive index – RI increases as protein concentration increases

18. Measurement of Adsorption of Radiation Infra-Red: protein absorb IR naturally due to characteristic vibrations of certain chemical groups NMR: measuring the area under a peak in an NMR chemical shift spectra that corresponds to the protein fraction

19. Measurement of Scattering of Radiation Light scattering: use degree of turbidity to measure protein content Ultrasonic scattering: ultrasonic velocity and absorption are related to concentration of protein aggregates present.

20. Advantages: Non-destructive Little or no sample preparation Rapid and precise Disadvantages: Calibration curve must be prepared Can only be used to analyze foods with relatively simple compositions Difficult to disentangle the contribution that the protein makes to the overall measurement from that of the other component

21. Protein Separation and Characterization Why need to have a knowledge of the effects of environmental conditions on protein structure and interaction? 1.It helps to determine the most suitable conditions to use to isolate particular protein from mixture of protein 2.Not select conditions which will not adversely affect the molecular structure of the protein

22. A. Methods Based on Different Solubility Characteristics Protein can be selectively precipitated or solubilized by altering pH, ionic strength, dielectric constant or temperature of a solution The most simple to use when large quantities of sample are involved

23. 1. Salting Out Proteins precipitate from aqueous solutions when the salt concentration exceeds a critical level Salt commonly used: Ammonium sulfate A two-steps procedure: Salt is added just below that necessary to precipitate out interest protein. Salt concentration is increased to point just above that necessary to cause precipitation

24. 2. Isoelectric Precipitation Isoelectric point (pI): pH where the net charge on the protein is zero Proteins tend to precipitate at their pI because there is no electrostatic repulsion keeping them apart Different amino acid has different pI.

25. 3. Solvent Fractionation ↓ in dielectric constant will ↑ magnitude of the electrostatic interaction between charged species Therefore, decrease the solubility of protein in solution Dielectric constant can be lowered by adding water-soluble organic solvents such as ethanol or acetone

26. 4. Denaturation of Contaminating Proteins To isolate protein that stable at high temperature or at extremes of pH Proteins are denatured and precipitated when heated at high temperature or in the high acid solution

27. B. Methods based on adsorption characteristics Involves the separation of compounds by selective adsorption-desorption at a solid matrix that is contained within a column Separation can be carried out using either an open column or high-pressure liquid chromatography

28. 1.Ion Exchange Chromatography Relies on the reversible adsorption-desorption of ions in solution to a charged solid matrix or polymer network Positively charged is called anion – exchanger while negatively charged is called cation – exchanger Two types of column are used 1.To bind protein of interest to the ion-exchange column 2.To favor adsorption from the column

29. 2. Affinity Chromatography Uses stationary phase that consists of a ligand covalently bound to a solid support The protein of interest binds to the ligand in the column, and eluted using another buffer solution

30. C. Methods Based on Size Differences Depends on the Stokes radius of a protein Stokes radius is the average radius that a protein has in solution Stokes radius increases in the following order: compact globular protein < flexible random-coil < rod-like protein

31. 1.Dialysis Use semi-permeable membranes that permit the passage of molecules smaller than a certain size through A protein solution is placed in dialysis tubing which is sealed and placed into a large volume of water or buffer which is slowly stirred LMW solutes flow through the bag while HMW remains.

32. 2. Ultrafiltration A protein solution is placed in a cell containing a semi-permeable membranes and pressure is applied to speed up the separation LMW protein pass through the membrane whereas the LMW molecules remains in the solution Use to concentrate a protein solution, remove salt, exchange buffers or fractionate protein on the basis of their size

33. 3. Size Exclusion Chromatography Also known as gel filtration Column which is packed with porous beads made of a cross-linked polymeric material Molecules larger than the pores in the beads are excluded and move quickly through the column whereas the movement of molecules which enter the pores is retarded

34. D. Separation by Electrophoresis Relies on differences in the migration of charged molecules in a solution when an electrical field is applied across it It can be used to separate protein on the basis of size, shape or charge

36. 1.Non-denaturing Electrophoresis A buffered solution of native proteins is poured onto a porous gel (usually polyacrylamide, starch or agarose) and a voltage is applied across the gel Gels may be contained between two parallel plates or in cylindrical tubes

37. 2. Denaturing Electrophoresis Separate protein based on their molecular weight Proteins are denatured prior to analysis by mixing them with mercatoethanol and sodium dodecyl sulfate (SDS) Smaller protein moving more rapidly through the matrix than larger molecules Type of electrophoresis commonly used is sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE)

38. 3.Isoelectric Focusing Electrophoresis Protein are separated by charge on a gel matrix which has a pH gradient across it Has the highest resolution selection of suitable gel is important

39. 4. Two Dimensional Electrophoresis Isoelectric focusing and SDS-PAGE are used together to improve resolution of complex protein mixture Proteins are separated in one direction on the basis of charge using isoelectric focusing and then in a perpendicular direction on the basis of size using SDS-PAGE

41. E. Amino Acid Analysis To determine amino acid composition of proteins A protein sample is first hydrolyzed to release amino acids which are then separated using chromatography