Tiêu đề 13. DEFINITION, CLASSIFICATION, CHEMISTRY AND METHOD OF ANALYSIS OF PROTEIN.pdf ✅

PHARMD GURU Page 1 DEFINITION: Proteins are the complex organic nitrogenous substances found in plants and animals. The term protein is derived from Greek word ‘proteios’ meaning „first‟. It was proposed by Mulder in 1838. INTRODUCTION TO PROTEIN:  Protein was first described by the Dutch chemist Gerhardus Johannes Mulder and further named by the Swedish chemist Jons Jakob Berzelius in 1838.  The term protein, derived from the Greek proteios, meaning first, are a class of organic compounds that are present in and vital to every living cell.  Proteins are large biochemical compounds (carbon, hydrogen, oxygen, and nitrogen) consisting of one or more polypeptides (amino acid residue) typically folded into a globular or fibrous form in a biologically functional way.  A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues i.e. an amine group (NH2), a carboxylic acid group (R–C=O–OH) and a side- chain (usually denoted as R) (Fig. 5.16).  They required for the structure, function, and regulation of the body‟s cells, tissues, and organs. Examples: Hormones, enzymes. DEFINITION, CLASSIFICATION, CHEMISTRY AND METHOD OF ANALYSIS OF PROTEIN
PHARMD GURU Page 2 They are stored in the form of aleurone grains in plants and can be extracted easily. They are purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation, electrophoresis, and chromatography techniques. CHEMISTRY:  The length of proteins and complexity vary based on the number and type of amino acid chains. There are about 20 different amino acids, each with a different chemical structure and characteristics; for instance, some are polar, others are non-polar. The final protein structure is dependent upon the composition of amino acids.  They consist of two polypeptide chains, a long chain which is on the left side, consisting of 346 amino acids and a short chain which is on the right side having 99 amino acids. The long chain is also known as heavy chain that contains 5 domains. In that 3 are extracellular domains (N1, C1 and C2) and a transmembrane domain where the polypeptide chain passes through the cell and a cytoplasmic domains (C-terminal) within the cytoplasm of the cell.  They are hydrolysed with acids or enzymes and break into amino acids.  They form colloidal solution in water.  Proteins are amphoteric in nature and get easily denatured due to heat, changes in pH, reaction with organic solvents etc. BIOCHEMICAL IMPORTANCE:  Proteins are the main structural and functional component of cytoskeleton. They are the main source of replacement of nitrogen in the body.  Proteins act as biocatalyst. They are enzymes.  Proteins are immunoglobulins that serve as first line defence against bacteria.  Structural proteins provide mechanical strength to the body.
PHARMD GURU Page 3  Storage proteins bind with specific substances and are stored in the body. Example, iron is stored in body as ferritin.  Transport proteins carry out the function of transporting specific substances either across the membrane or in the body fluids. CLASSIFICATION OF PROTEINS: Broadly proteins are classified in three types tabulated in Fig. 5.17.
PHARMD GURU Page 4 ISOLATION OF PROTEINS: Various modern isolation techniques are used for the isolation of proteins that include cell disruption method, mechanical homogenization, solid-phase micro- extraction, supercritical-fluid extraction, pressurized-liquid extraction, microwave- assisted extraction, solid-phase extraction and surfactant-mediated techniques. Generally, protein is extracted with trichloroacetic acid (TCA) and acetone or by using phenol or by homogenization with buffer. Plant tissue is homogenized in 10% TCA containing 2% beta mercaptoethanol using liquid nitrogen. Further the solution is kept at –20°C for overnight to form the precipitate which is subjected to centrifugation at high r.p.m for 30-40 minutes at 4°C. The precipitate is washed with cold acetone. ANALYSIS METHODS OF PROTEINS: 1. BIURET METHOD:  A violet-purplish colour is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions.  The biuret reagent is mixed with a protein solution and then allowed to stand for 15-30 minutes and then the absorbance is taken at 540 nm. 2. LOWRY METHOD:  The lowry method combines the biuret reagent with another reagent i.e. Folin- Ciocalteau phenol reagent which reacts with tyrosine and tryptophan residues in proteins.  This gives a bluish color which can be read somewhere between 500-750 nm.  There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. 3. TURBIMETRIC METHOD: Protein molecules which are normally soluble in solution are precipitated by the addition of certain chemicals, e.g., trichloroacetic acid. Protein precipitation causes the solution to become turbid and the final concentration of protein is determined by measuring the degree of turbidity.