Protein Denaturation (HNU Section 5) PDF

Medical laboratory program Faculty of health sciences technology-HNU (2024-2025)  What is protein??? High molecular weight , N2 containing organic compound present in all types of cells. Have several cellular functions. Enzymes Hormones...

Medical laboratory program Faculty of health sciences technology-HNU (2024-2025)  What is protein??? High molecular weight , N2 containing organic compound present in all types of cells. Have several cellular functions. Enzymes Hormones Receptors Insulin Insulin receptors Structural proteins Source of energy Phosphoproteins (protein degradation to amino acids)  Structure of proteins – Classification Building unit (amino acid) 1- Simple proteins Prosthetic gp Albumin , Globulin 2- Conjugated protein Phosphoproteins, Glycoproteins Undergo hydrolysis 3- Derived protein Amino acids, Peptides, peptones Protein Properties of proteins s  Proteins Properties of proteins ✓ Due to presence of (COOH) & (NH2) gps , they can carry both –ve & +ve charges ✓ These charges stabilize the protein in the soln (dispersed) → prevent ppt. ✓ Amphoteric nature (act as acid + act as base) Folding of protein structure aids dispersion + water shell Hydrophilic Hydrophobic The Net charge of the protein is affected by pH. High PH Low PH (de-protonation) (protonation) Remove H+ from Add H+ on NH2 gp +ve = -ve COOH gp Net charge= zero Soluble Soluble Precipitate  Pptn. of AA in diff. media +Ve -Ve Net Charge (ZERO)  Determination of PI for milk casein Principle Ability of dissolved protein (casein of milk) at its pI to ppt → cloudy white soln. Set up a series of buffers with varying pH Note the pH at which precipitation occurs. This pH is the isoelectric point (pI) of casein. Protein denaturation  Structural Organization Folding Hydrophilic 1ry Hydrophobic Native protein 2ry 3ry Biologically 4ry Biologically active inactive  Structural Organization Folding Hydrophilic 1ry Hydrophobic Unfolding & disruption of native protein conformation Native protein 2ry 3ry Biologically 4ry Biologically active inactive Not affected Physical Chemicals Heat, UV Organic solvents  Behavior of denatured protein Hydrophobic core Hydrophilic surface DENATURED Denaturing agents Folded protein Unfolding …>forces some hydrophobic AA to surface AGGREGATED Denaturating agent Conc Mineral Acids Organic Acids Heavy Metal Salts Organic Solvent  Pptn. of AA in diff. media -Ve +Ve salts salts TCA , +Ve Zn2+ , Ba2+ , Phosphotungestate -Ve Cd2+ & Cu2+ , perchlorate Net Charge &sulfosalicylate (ZERO) PPt PPt Denaturation by Conc mineral acids Principle Conc. HCL or HNO3 Cause charge neutralization Destruction of protein spatial structure PPT of protein  Denaturation by Organic solvent Principle Chloroform – ethanol - acetone Destroy hydrophobic interaction within protein molecule + disrupt solvation layer, decreasing hydration. Destruction of protein spatial 3D structure PPT of protein  Denaturation by Organic acids Principle Trichloroacetic acid Both Mechanisms Acid organic moiety Neutralize charge destroy hydrophobic PPT of protein interaction  Trichloroacetic acid TCA organic moiety Acid destroy Neutralize charge hydrophobic interaction PPT of protein destroying hydrophobic interaction “hydrophobic gp”  Denaturation by heavy metals Pb+2 Pb+2 S Pb+2 SH S SH S SH SH S Pb+2 Pb+2 Pb+2 Pb+2 EXCESS Pb+2 Pb+2 Pb+2 Pb+2 Pb+2 Pb+2 SH Pb+2 SH SH SH Pb+2 Pb+2 Pb+2 renaturation Pb+2 SH SH Pb+2 Pb+2 Pb+2 Pb+2 SH Pb+2 SH Pb+2 Pb+2 ppt Pb+2 protein denaturation Procedures : A- protein denaturation : 0.5 ml milk+ 0.5 ml HCL observe 0.5 ml milk + 1ml Acetone observe 0.5 ml milk + 0.5 ml TCA observe 0.5 ml milk + 0.5 ml CuSo4 observe add xss Cuso4 observe B- Determination of isoelectric point of milk Casein: 1 ml milk + 1 ml of different buffers ( PHs 2, 4.6, 9) observe  Proteins Denaturation Denaturing Observation Mechanism of agent denaturation Mineral Acid: Neutralize charge PPT of protein (HCl) Organic Solvent: destroy hydrophobic interaction PPT of protein (acetone) Organic Acid: Acid : neutralize charge PPT of protein Organic moiety destroy hydrophobic (TCA) interaction Heavy Metal PPT of protein Salt: bind to the sulfur of the side (cuso4) And redissolve after chain of amino acids, causing destruction of bi adding exss cuso4 sulphid bond leading to denaturation and precipitation of the protein. Resolvation due to new charge  Determination of Isoelectric Point pH Observation Comment 2 No change pHpI Cause deprotonation of protein (soluble) Thank you