BCH 201 General Biochemistry I Methods of Protein Isolation, Purification, and Identification PDF

BCH 201-GENERAL BIOCHEMISTRY I Methods of Protein isolation, purification and identification BY DR DERE T.O OUTLINE Introduction Methods of isolation of these proteins; Purification of proteins Quantification of the various protei...

BCH 201-GENERAL BIOCHEMISTRY I Methods of Protein isolation, purification and identification BY DR DERE T.O OUTLINE Introduction Methods of isolation of these proteins; Purification of proteins Quantification of the various proteins. Effects of acids and alkalis on proteins; INTRODUCTION Proteins are the biomolecules composed of amino acid, forming the building block of the system and performs most of the biological functions of the system. The major role is to provide the body's building blocks. They are the precursors of several biologically relevant molecules. Therefore, either the excess or deficiency of protein can lead to disease, resulting in nervous system defects, metabolic problems, organ failure, and even death. ISOLATION OF PROTEINS The isolation of the peptide product can be improved by applying method development principles that are often associated with analytical high pressure liquid chromatography (HPLC). The separation mechanisms used to create a purification method at the larger preparative scale are the same as those principles that are utilized at the smaller scale. Column selection, choice of mobile-phase modifier, the use of temperature, and gradient optimization are choices that must be examined in the development of any separation method. Systematic method development for peptide isolations can improve the purity and yield of product significantly. PURIFICATION OF PROTEIN PURIFICATION OF PROTEIN PURIFICATION OF PROTEINS A. PURIFICATION BASED ON MOLECULAR SIZE 1. Dialysis and ultrafiltration 2. Density gradient centrifugation 3. Size-exclusion chromatography 1. DIALYSIS AND ULTRAFILTRATION 2. DENSITY GRADIENT CENTRIFUGATION 3. SIZE EXCLUSION CHROMATOGRAPHY 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D TYPES OF CHROMATOGRAPHY A. Liquid chromatography B. Gas chromatography C. Paper chromatography D. Thin layer chromatography E. Column chromatography F. Gel-filtration chromatography G. Affinity chromatography H. High pressure liquid chromatography(HPLC) 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D 3. SIZE EXCLUSION CHROMATOGRAPHY CONT’D B. BASED ON SOLUBILITY OF PROTEIN 1. Salting out 2. Iso-electric precipitation 1. Salting out 1. Salting out cont’d 2.Iso-electric precipitation 2.Iso-electric precipitation cont’d C. BASED ON ELECTRIC CHARGE 1. Ion- exchange chromatography 2. Electrophoresis 1. Ion- exchange chromatography 1. Ion- exchange chromatography cont’d 2. Electrophoresis 2. Electrophoresis cont’d 2. Electrophoresis cont’d 2. Electrophoresis cont’d 2. Electrophoresis cont’d 2. Electrophoresis cont’d  SUMMARY Clinical relevance Clinical relevance Clinical relevance Clinical relevance Clinical relevance  QUANTIFICATION OF VARIOUS PROTEINS METHODS OF QUANTIFICATION OF PROTEINS 1.- BRADFORD OR COOMASIE BLUE TEST 2.- LOWRY TEST 3.- BICINCHONINIC ACID (BCA) 4.- BIURET METHOD 5.- ULTRAVIOLET (UV) ABSORPTION 1.- BRADFORD OR COOMASIE BLUE TEST Concentration range: 20-2000 ug/ml Characteristics: Method described in 1976. Negatively charged Coomasie blue stain binds positively charged proteins. Binds to arginine, tryptophan, tyrosine, histidine and phenylalanine residues. Staining in solution is red and absorbs at 465 nm, while when bound to the basic amino acids of a protein it turns blue and absorbs at 595 nm. The absorbance measurement is compared to the values of a standard curve to determine the protein concentration of the sample. 2.- LOWRY TEST Concentration range: 10-1000 ug/ml. Characteristics: One of the most used methods to quantify proteins, was developed in 1951. The reaction occurs in two steps: Formation of Cu-N complexes (present in the protein). Tyrosine and tryptophan complexes react with Folin -Ciocalteau reagent, producing a blue-green colour that absorbs between 650 and 750 nm. The absorbance measurement is compared to the values of a standard curve to determine the protein concentration of the sample. 3.- BICINCHONINIC ACID (BCA) Concentration range: 20-2000 ug/ml Characteristics: Developed in 1985. Colorimetric test where the absorbance will be proportional to the protein concentration present in the sample. As well as in the Lowry test, a reaction occurs in two steps: Formation of protein-copper ion complexes. Formation of a Cu-BCA chelate that gives rise to an intense purple coloration that absorbs at 562 nm 4.- BIURET METHOD Concentration range: 0.373-80 g/L Characteristics: It is based on the formation of a coloured complex between the Cu2+ and the NH groups of the peptide bonds in a basic medium. 1Cu2+ complexes with 4 NH. The colour intensity is directly proportional to the amount of protein (peptide bonds) and the reaction is quite specific, so few substances interfere. The absorbance of the colour produced is read at 540 nm. The sensitivity of the method is very low and it is only recommended for the quantification of proteins in highly concentrated preparations (for example, in serum). The Biuret reagent is prepared as follows: – Dissolve 3.8 g of CuSO4.5H2O and 6.7 g of NaEDTA in 700 ml of H2O. – While stirring add 200 ml of 5N NaOH and then 1 g of KI as stabilizer. – Store in a plastic bottle. 5.- ULTRAVIOLET (UV) ABSORPTION Concentration range: 0,1-100 ug/ml. Characteristics: By measuring the characteristic absorption of tryptophan and tyrosine at 280 nm, this method estimates the amount of protein present in the sample. Advantages: – Fast and relatively sensitive method.  EFFECTS OF ACID AND ALKALIS ON PROTEIN Effects of Acid and Alkalis on protein Proteins are large molecules found in our bodies and food, consisting of many smaller components called amino acids. Proteins have the properties they do because of the shape and arrangement of their amino acids. A weak bond, known as a hydrogen bond, forms between a hydrogen atom and an oxygen atom in the amino acids. This gives the protein its shape. Effects of Acid and Alkalis on protein What is denaturing and how does it happen? A protein becomes denatured when its normal shape gets deformed because some of the hydrogen bonds are broken. Weak hydrogen bonds break when too much heat is applied or when they are exposed to an acid (like citric acid from lemon juice). As proteins deform or unravel parts of structure that were hidden away get exposed and form bonds with other protein molecules, so they coagulate (stick together) and become insoluble in water. Effects of Acid and Alkalis on protein Use of lemon and lime juice is an example of protein acid denaturation. Explore -Place an egg white into a clean bowl -Observe the colour and texture of the egg white -Now add 3ml of lemon juice to the egg white and stir Record what happens to the colour and texture of the egg white Egg white turns solid and goes white instead of clear when it denatures  THANKS FOR YOUR ATTENTION