lecture 8 for student

1 SPECTROSCOPY PROTEIN DETERMINATION PHAM VAN HUNG, PhD Introduction  Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure.  Component?  Amino acids?  Classification?  Structure?  Functional properties? Primary Structure of Protein Secondary Protein Structure Quaternary structure  A protein has size and shape as well as unique arrangement of its polypeptide chains. (Aggregation of several peptide chains to form a definite molecule by ionic bond, hydrogen bond, and/or hydrophobic bond). 2 SPECTROSCOPY Importance of protein analyses  Protein analysis is important for:  Nutrition labeling  Pricing  Functional property investigation  Biological activity determination Importance of protein analyses  Protein analysis is required when you want to know: 1. Total protein content 2. Content of a particular protein in a mixture 3. Protein content during isolation and purification of a protein 4. Nonprotein nitrogen 5. Amino acid composition 6. Nutritive value of a protein Protein Determination Methods 1. Kjeldahl Method. 2 Dye Binding Method. 3. Biuret Method. 4. Lowry Method. 5. Ultraviolet Method. Kjeldahl Method - Nitrogen Determination (1) Digestion to conver nitrogen into an ammonium ion (NH 4 + ). + conc. H 2 SO 4 + a catalyst (Copper sulfate) (2) Neutralize NH 4 + with NaOH to get NH 3 (3) Steam distillation of NH 3 and trap in boric acid. (4) Titrate with hydrochloric acid. Calculation: Gram nitrogen/ gram of sample = *(ml of sample - ml of blank) × N (normality) of standard acid × 0.014g/meq weight of sample * ml of hydrochloric acid required to titrate sample solution. Procedure and reactions  Sample preparation  Digestion  Neutralization and Distillation  Titration  Calculations [2] [5] Conversion Factors from Nitrogen to Protein for Foods Corns Milk Whole wheat Wheat flour Nuts Eggs Barley Peas Oats Meat Rye 6.25 6.38 5.83 5.70 5.30 Beans Millet 3 SPECTROSCOPY Apparatus Apparatus Apparatus Application Advantages: 1. Applicable to all types of foods 2. Inexpensive (if not using an automated system) 3. Accurate; an official method for crude protein content 4. Has been modified (micro Kjeldahl method) to measure microgram quantities of proteins Disadvantages: 1. Measures total organic nitrogen, not just protein nitrogen 2. Time consuming (at least 2 h to complete) 3. Poorer precision than the biuret method 4. Corrosive reagent Dye Binding Method Principle: - At low pH, basic groups of protein are (+) charged. These will quantitatively bind a (-) charged dye. - Proteins bind the dye to form an insoluble complex. The unbound soluble dye is measured after equilibration of the reaction and the removal of insoluble complex by centrifugation or filtration. NH 3 + CH CH 2 CH 2 CH 2 CH 2 N N H H C O C CH 2 C NH + CH CH N HC H C O N H CH 2 CH 2 CH 2 N H C NH 2 NH 2 + Lysine Arginine Histidine Acid Orange 12: N = N HO SO 3 - Procedure: 1. Mix protein, dye, buffer pH = 2. 2. Filter or centrifuge. 3. Measure absorbance of filtrate. Dye Binding Method 4 SPECTROSCOPY Absorbance of dye bound by protein = A initial (free) die concentration - A. filtrate die concentration Dye Binding Method A b s o r b a n c e a t 4 7 0 n m % Protein (Kjeldahl) 6 8 10 12 14 16 Skim milk 1 1 2 2 x x x x Factors Influencing Dye Binding determination: 1. Temperature 2. Non-proteins. 3. Buffers systems. 4. Protein quality. Dye Binding Method Biuret Method Principles: Cu ++ in alkaline solution form complexity with peptide bonds - give pinkish-purple color. Measure the intensity of color at 540 nm. Standard is bovine serum albumin (BSA). A a t 5 4 0 n m % Protein (Kjeldalh) Lowry Method The Lowry method combines the biuret reaction with the reduction of the Folin–Ciocalteau phenol reagent (phosphomolybdic- phosphotungstic acid) by tyrosine and tryptophan residues in the proteins. •Cu ++ in alkaline solution to form complexity with protein. •Cu ++ catalyses oxidation of phenol group of tyrosine with phosphomolybdic-phosphotungstic acid. Procedure 1. Proteins to be analyzed are diluted to an appropriate range (20–100 μg). 2. K Na Tartrate-Na2CO3 solution is added after cooling and incubated at room temperature for 10 min. 3. CuSO4-K Na Tartrate-NaOH solution is added after cooling and incubated at room temperature for 10min. 4. Freshly prepared Folin reagent is added and then the reaction mixture is mixed and incubated at 50◦C for 10 min. 5. Absorbance is read at 650 nm. 6. A standard curve of BSA is carefully constructed for estimating protein concentration of the unknown. Application Advantages: 1. Very sensitive  (a) 50–100 times more sensitive than biuret method  (b) 10–20 times more sensitive than 280-nm UV absorption method 2. Less affected by turbidity of the sample. 3. More specific than most other methods. 4. Relatively simple; can be done in 1–1.5 h. Disadvantages: 1. Color varies with different proteins to a greater extent than the biuret method. 2. Color is not strictly proportional to protein concentration. 3. The reaction is interfered with to varying degrees by sucrose, lipids, phosphate buffers, monosaccharides, and hexoamines. 4. High concentrations of reducing sugars, ammonium sulfate, and sulfhydryl compounds interfere with the reaction. 5 SPECTROSCOPY Ultra-violet Absorption (UV) at 280 nm 1. Proteins show strong absorption in the region at ultraviolet (UV) 280nm, primarily due to tryptophan and tyrosine residues in the proteins. Chromophoric side chains of aromatic amino acids (Tyrosine, Tryptophan). 2. Because the content of tryptophan and tyrosine in proteins from each food source is fairly constant, the absorbance at 280nm could be used to estimate the concentration of proteins, using Beer’s law. Procedure 1. Proteins are solubilized in buffer or alkali. 2. Absorbance of protein solution is read at 280nm against a reagent blank. 3. Protein concentration is calculated according to the equation A = ε lc where: A = absorbance ε = absorptivity l = cell or cuvette path length c = concentration Determination for Amino Acid Compositions of Proteins A. Hydrolysis 1. Overnight in 6 M HCl at 100 C. 2. Enzymes. B. Separation by ion exchange chromatography. Mechanism of Ion-Exchange Chromatography of Amino Acids Na + Na + H + OH - = H 2 O = H 2 O OH - H + Na + Na + COO - H 3 N + Na + OH COO - N + H 3 H 3 N + COOH OH COOH COOH H 3 N + So 3 - SO 3 - SO 3 - So 3 - So 3 - SO 3 - H 3 N + Exchange Resin pH 2 pH3.5 pH4.5 LYS HIS ASP GLU ALA VAL LEU pH 2.25 pH 3.25 pH4.25 Moles/Liter Chromatogram of Amino Acids The end! . Factors from Nitrogen to Protein for Foods Corns Milk Whole wheat Wheat flour Nuts Eggs Barley Peas Oats Meat Rye 6.25 6. 38 5 .83 5.70 5.30 Beans Millet 3 SPECTROSCOPY. incubated at room temperature for 10 min. 3. CuSO4-K Na Tartrate-NaOH solution is added after cooling and incubated at room temperature for 10min. 4. Freshly