Protein Analysis Lecture - King Abdul-Aziz University PDF

King Abdul-Aziz University Faculty of Applied Medical Sciences Department of Clinical Nutrition Food Science 2 (CLN 221) Protein Analysis Dr. Leila Arfaoui 18/3/2025 1  Learning Outcomes After atte...

King Abdul-Aziz University Faculty of Applied Medical Sciences Department of Clinical Nutrition Food Science 2 (CLN 221) Protein Analysis Dr. Leila Arfaoui 18/3/2025 1  Learning Outcomes After attending this lecture, you will be able to: 891,3 w̅ ❑ justify the utility of protein analysis ❑ explain the principle of the different techniques used for protein extraction and analysis ❑ outline the limits of the different techniques used for protein extraction and analysis Dr. Leila Arfaoui 2  Introduction ◼ Proteins are polymers of amino acids. ◼ Major structural components of many natural foods. ◼ Food analysts and dietitians are interested in knowing the total concentration, type, molecular structure and functional properties of proteins in foods. Dr. Leila Arfaoui 3  Protein Analysis Importance 1. Legal: to conform food regulations like lipids 2. Nutrition labeling 3. Health: nutritional importance (major source of energy, essential amino- acids: lysine, methionine, leucine, isoleucine…) 4. Quality: Functional properties investigation Isolated proteins are often used in foods as ingredients for their functional properties. e.g. gliadin and glutenine in wheat flour for bread making (dough elasticity), casein in milk for coagulation into cheese products, and egg albumen for foaming, gelling agents (gelatin) Dr. Leila Arfaoui 4  Determination of Protein Concentration 1. Kjeldahl Method Jw ◼ A food digested with strong acid so that it releases nitrogen which can be determined by a suitable titration technique. ◼ The process involves: (1) digestion with concentrated sulfuric acid to convert organic nitrogen into e ammonium sulfate (2) 25 11 43 neutralization with a strong base (e.g., NaOH) and distillation to release ammonia Gas NH ftp.III It (3) 2 titrated with acid (HCl) to determine nitrogen content. Dr. Leila Arfaoui 5  Kjeldahl Method ▪ Does not measure the protein content directly. do Total Nitrogen(%)= (V×N×1.4007)​/W we ✓ V = Volume of acid (HCl) used for titration (mL) ✓ N = Normality of the acid warof ✓ 1.4007 = Conversion factor for nitrogen warm i ii sii ✓ W = Sample weight (g) ▪ Conversion factor is needed to convert the measured nitrogen concentration to a protein concentration. Protein Content(%)=Total Nitrogen (%)×F A conversion factor (F) of 6.25 is commonly for general proteins, but varies for different food types (equivalent to 0.16 g nitrogen per gram of protein) Dr. Leila Arfaoui 6  Kjeldahl Method ii ◼ Advantages: ◼ Disadvantages: ❖ universal ❖ does not give a measure of ai ❖ high precision true protein. ❖ good reproducibility ❖ different proteins need Jss d I different correction factors. ❖ the use of concentrated sulfuric acid at high temperature. hazard ❖ time consuming. Dr. Leila Arfaoui 7  Dumas Method 2. Dumas Method ◼ Principle: a sample of known mass is combusted in a high temperature to release CO2, H2O and N2. soya s.jo ◼ Nitrogen content is measured by separating N2 from CO2 and H2O using a column (gas chromatography) ◼ Also used conversion factor to determine protein content. protein Jence a conversion factor Dr. Leila Arfaoui 8  Dumas Method ◼ Advantages ◼ Disadvantages ❖ Faster ❖ High initial cost ❖ Doesn’t need toxic chemicals ❖ Does not give a measure of or catalysts true protein ❖ Easy to use ❖ Small sample size make its ❖ Samples can be measured difficult to obtain a automatically representative sample o wow Is a if I by toys L a g Dr. Leila Arfaoui 9  spectrophotometer egw.IM UV-Visible Spectroscopy a.hr a'www.fjj INgIw1gjiois.li 1 1 3. Methods using UV-Visible Spectroscopy ◼ in Use 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 ◼FL. m built first. Main difference: the chemical groups which are responsible for the m absorption or scattering of radiation. is Dr. Leila Arfaoui 10 Calibration curve of absorbance versus protein concentration Dr. Leila Arfaoui 11  UV-Visible Spectroscopy A. Direct Measurement at 280 nm ◼ Principle: tryptophan and tyrosine absorb ultraviolet light strongly at 280 I.to's www nm iwiwobfcfji ◼ Use the same wavelength to measure protein concentration ◼ Advantages: simple to carry out, non-destructive and no special reagents are required w.i mrisgyja.is RNADNA adown ◼ Disadvantages: nucleic acid absorbs light at 280 nm To overcome: nucleic acids absorbs light also at 260 nm. So, it they can be corrected using a method based on the absorption difference between 260 and 280 nm. 1260 Proteinconcentration Ao Dr. Leila Arfaoui 12  UV-Visible Spectroscopy ▪ Since nucleic acids contribute to absorbance at 280 nm, their interference is corrected using absorption differences at 260 nm and 280 nm. Dr. Leila Arfaoui 13 ingo 8t.W.ws 911 UV-Visible Spectroscopy Now Carte dos I Method B. Biuret ◼ 55 FI I ÉI1gW at A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions to ◼ Absorbance is read at 540 nm ◼ Advantages: no interference from materials that absorb at lower w/lengths w.gg we glad 5 300200m w̅ RNADNA a d ◼ Disadvantages: low sensitivity compared to Lowry method Dr. Leila Arfaoui 14  UV-Visible Spectroscopy C. Lowry Method now.si plso1 Cas issdass sa.windpi. ◼ Combines Biuret reagent and Folin-Ciocalteau phenol reagent which 4w.esya reacts with tyrosine and tryptophan residues in proteins. ◼ gjicolor which can be read at w/length between 500 – 750 nm Gives a bluish ◼ More sensitive to low concentrations of protein than Biuret method Dr. Leila Arfaoui 15  Turbidimetric Method isaaye wso.ms o.ws ig precipitate ◼ Proteins form by addition of certain chemical e.g. trichloroacetic acid. x̅ T.is Isi ◼ The concentration of protein is determined by measuring the degree of turbidity. ON8165582415 ◼ The turbidimeter directs a beam of monochromatic light (often in MIT ji Is v80 s the infrared or visible range) through the sample. Scattered light Toolog.at His intensity is measured and it is proportional to the concentration of precipitated proteins. ‫ف‬ Dr. Leila Arfaoui 16  Turbidimetric Method ▪ Standard curve turbidity versus protein concentration should be prepared using a series of protein solutions of known concentration Advantages: Disadvantages: ◼ Rapid ◼ difficult to quantitatively extract ◼ Simple to carry out proteins from certain types of ◼ Sensitive to low protein food e.g. processed food concentration B ah Dr. Leila Arfaoui 17  Protein Separation and Characterization ◼ Why need to have a knowledge of the effects of environmental conditions on protein structure and interaction? a.ws04 Is ewl.PH ow 1. It helps to determine the most suitable conditions to use to isolate particular protein from a mixture of protein 2. Avoid selecting conditions that can adversely affect the molecular structure of the protein II 1__I1 Dr. Leila Arfaoui 18  Protein Separation and Characterization 1. Methods Based on Different Solubility Characteristics ◼ Protein can be selectively precipitated or solubilized by altering pH, ionic strength, or temperature of a solution. ◼ The simplest to use when large quantities of sample are involved. Dr. Leila Arfaoui 19  Protein Separation and Characterization 1. Methods Based on Different Solubility Characteristics oigdswo.TW a. Salting Out EW LI sG ◼ Proteins precipitate from jgjlul.gg aqueous solutions when the salt concentration exceeds a critical level. ◼ Salt commonly used: Ammonium sulfate. and Kjeld Dr. Leila Arfaoui 20  Protein Separation and Characterization www.II s.dJ b. 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. 81 4331 at d a issues s 1,15 2 ◼ Different amino acid has different pI. Dr. Leila Arfaoui 21  Protein Separation and Characterization andV IN a 11Aj's c. Denaturation of Contaminating Proteins ◼ To isolate protein that is stable at high temperature or at extremes of pH. 15950.531504 I ◼ Proteins are denatured and precipitated when heated at high temperature or in the high acid solution. Dr. Leila Arfaoui 22  Protein Separation and Characterization 2. Methods Based on Size Differences Mla jigs ma ◼ Depends on the Stokes radius of a protein (in solution) ◼ Stokes radius is the average radius that a protein has in solution. Dr. Leila Arfaoui 23  Protein Separation and Characterization a. Dialysis jwe.si I n ◼ 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. ftp.jsdsw.ca.jdjawtokgilsyw1 sba.d.gl ◼ LMW solutes flow through the bag while HMW remains. low idar Tighmolecular weight jitsu Dr. Leila Arfaoui 24 Dialysis Tubing Dr. Leila Arfaoui 25  Protein Separation and Characterization b. Ultrafiltration IWAI ▪ A protein solution is placed in a cell containing a semi-permeable membranes and pressure is applied to speed up the separation. dialysisse ▪ LMW protein pass through the membrane whereas the HMW molecules remains in the solution. 04 6 b ssp.dl.it ▪ Use to concentrate a protein solution or fractionate protein on the basis of their size. I s Dr. Leila Arfaoui 26  Protein Separation and Characterization 3. Methods based on adsorption and electric characteristics: ◼ Separation can be carried out using high-pressure liquid chromatography (HPLC). ◼ 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. Dr. Leila Arfaoui 27  Amino Acid Analysis To determine amino acid composition of proteins: 424 as 100 Dss saw 1 i wussy ◼ A protein sample is first hydrolyzed to release amino acids which are then separated using chromatography. Johexchange chromatography IPLC Dr. Leila Arfaoui 28  Comparison between the different methods Sample preparation: The Kjeldahl, Dumas, and UV spectroscopy methods require little preparation. While the other methods require very fine particles for extraction of proteins from the complex food systems Principle: The Dumas and Kjeldahl methods measure directly the nitrogen content of foods. However, the Kjeldahl method measures only organic nitrogen plus ammonia, while Dumas measures total nitrogen, including the inorganic fraction (Therefore, Dumas gives a higher value for products that contain nitrates/nitrites.) Sensitivity: Kjeldahl, Dumas, and Biuret methods are less sensitive than Lowry or UV methods. 40 500750 Dr. Leila Arfaoui 29  Comparison between the different methods Speed: the speed of determination in the colorimetric methods and in the Dumas method is faster than with the Kjeldahl method. Applications: Although both Kjeldahl and Dumas methods can be used to measure N content in all types of foods, in recent years the Dumas method has largely replaced the Kjeldahl method for nutrition labeling (since Dumas method is faster, has a lower detection limit, and is safer). However, the Kjeldahl method is the preferred method for high-fat samples/products since fat may cause an instrument fire during the incineration procedure in the Dumas method. Dr. Leila Arfaoui 30