Protein Purification and Characterization Techniques PDF

Chapter Five Protein purification and characterization technique Salting Out: Ammonium sulfate used for “salt out” :Function takes away by iterating with, it makes protein less soluble because of hydrophobic interactions among proteins.to get closer to 30,40,50,75 increments Column chromatography in...

Chapter Five Protein purification and characterization technique Salting Out: Ammonium sulfate used for “salt out” :Function takes away by iterating with, it makes protein less soluble because of hydrophobic interactions among proteins.to get closer to 30,40,50,75 increments Column chromatography includes 2 phases(it contains sodium chloride or potassium chloride) 1. Stationary phase:samples interacts with this phase metrics specific chemical components 2. Mobile:samples flows over the stationary phase and carries along with to be separated (several buffer solutions ) Affinity chromatography:access to mimic of molecular compound 1. Step 1:Column is packed with Stationary phase(s) 2. Step 2 :A mixture containing the target biomolecule is passed through the column (Binding) 3. Step 3 :Washing Non Target molecule that do not bind are washed away with buffer leaving the target molecule 4. Step 4: Elution:release by changing ph and ionic strength or using competition ligand to disrupt the binding interaction Ion Exchange :Cation/Anion Exchange What is it ? Cation exchange of positively charged ions between solution and a solid(Resin) How does it works? Resin is negatively which attracts and holds cations from the solution when cations in the solution come into contact with resin int they are exchange for other cations are already bonded to resin Anion Exchange: Exchange between anions and cations Example :If Na+ are removed from a solution they can be exchange for calcium ions (Ca2+) from the resin Calcium exchange: if chloride ions (cl-) are removed from a solution they can be exchange from hydroxide ions(OH-) from the resin Size Exclusion/Gel-filtration chromatography Smaller molecules can enter pores of a stationary phase while large molecules cannot,large molecules comes out first then smaller(weak interactions) Electrophoresis:Charge molecules migrate in electric field towards opposite charge The more the negative charge faster Proteins have different mobility:Charge and size(SDS treatment which is the smaller and negative Primary Structure Determination: Peptide Sequencing:Edman Degradation Protein Cleavage: Specific sites by 1.Enzymes(Trypsin at C-terminal of (+) charge side chains) ( chymotrypsin cleaves at C terminal of aromatics) 2.Chemical reagents (Cyanogen bromide) Determining Proteins Sequence Can be separated by chromatographic techniques Determination of Tertiary and Quaternary Structure:X-ray and NMR data It determines the 3 structures of proteins:mainly the Diffraction pattern produced by electrons scattering X-rays. Predicting Protein Folding: Bioinformatics: First step to predict structure is sequence homology(series of amino acids) Chapter Six and Seven Enzymes;Mechanism, behavior,regulation Enzyme Catalysis:They are inorganic enzyme proteins Example Platinum Surface the activation of energy kJmol is lower and for a catalyst is higher For the kcal mol for a inorganic catalyst is higher and lower for catalyst Catalytic Mechanism:Can be found at the levels of protein chain 1.General Acid-base Catalysis:donation and acceptance of protons it Mostly group C which are acidic Aspartic( Asp,D) and Glutamic Acid (Glu,E) And Group D polar basic : Histidine( His,H), Arginine(Arg,R) and Lysine (Lyst,K) 2.Nucleophilic Substitution Catalysts:Nucleophilic electron rich atom attacks electron deficient atom :Group B neutral polar(Serine Ser,S) Cysteine (Cys,C) and tyrosine(Thr,T) 3.Electrophilic Substitution Catalyst:Electrophilic electron poor atom which bonds electron deficient atom metal ions Chymotrypsin:is an enzyme that it function as a catalyst for a reaction hydrolysis breaking the peptide bonds and increasing the rate of reaction, it can be slower,depends on the rate of reaction, it target the peptide bonds at the C terminal level aromatic side chains, non covalent interaction Chymotrypsin:Effect of DIPF DIPF inactivates chymotrypsin by reacting with serine-195C Chymotrypsin:Effect of TPCK TPCK is a reactive group H57 is also critical for activation of enzyme it folds tertiary structure, no tertiary structure, no catalyst Coenzymes: They are small molecules,coreactant, non protein substance that takes part in an enzymatic reaction and is regenerated for further reaction They can be metal ions that behave as coordination compounds(Zn2+,Fe2+) And vitamins like NADH or NADPH 2 Modes of E-S complex Formation 1.Lock and key model:complementary to one another, mirror structure 2.Induced-fit model:is not too stable, undergoes a conformational change upon binding to substrate it becomes complementary only after the substrate binds to the enzyme Michaelis-Menten Kinetics Enzyme Inhibition: Reversible inhiibitor:A substance that binds to an enzyme to inhibit it but can be realized Irreversibele Inhibitor :A substance that causes inhibition that cannot be reversed Homotropic effects :Allosteric interactions that occur when direct substrates or products bind to the protein (Aspartate to ATCase) Heterotropic effects:Allosteric interactions that occur when different substances are bound to the protein inhibitor of ATCase by CTP and activation by ATP Two Types of Allosteric Enzymes 1.K system: a enzyme which an inhibitor or activators alters K0.5 2.V System:an enzyme for which an inhibitor or activator alters Vmax which is one-half Control of Enzyme Activity via Phosphorylation What is Phosphorylation?Specific side chains,covalent at the level of primary structure:ion ion, ion dipole, dipole dipole Chapter Eight Lipids and Proteins are Associated in Biological Membranes What is a lipid: is an organic compound, insoluble in water but soluble in aprotic organic solvents,amphipathic in nature. It includes fatty acids, triacylglycerols,phosphoacylglycerols. Steroids:Its a group of lipids that have fused-ring structure of 3 six membered rings and 1 five membered ring is a product of hydrocarbon saturated and unsaturated Fatty Acid: An unbranched-chain carboxylic acid they only contain c=c Unsaturated:Oleic Acid Saturated :Palmitic Acid,Stearic acid Unsaturated:linoleic acid,Arachidonic acid Triacylglycerol (triglyceride) :An ester of glycerol with three fatty acids Phosphoacylglycerols(Phospholipids): is a alcohol group of glycerol is esterified by phosphoric acid Lipids Bilayers: that are only fatty acids The polar surface contains charged groups And the hydrophobic tails lies in the interior of the bilayer Detergent :Long tail and fewer double bond Short tail high abundance of double bonds more flexible phospholipid Fluid Mosaic Model Fluid:lateral motion of components in the membrane Mosaic:Components in the membrane exist side by side separated entities structure of lipid bilayer with proteins glycolipids and steroid such as cholesterol, no complexes example lipid-protein complexes are formed Membrane Proteins Functions:transport substances across membrane,receptor sites and sites of enzyme catalysis, noncovalent, ion dipole, dipole dipole,loosely bonds 3 different categories 1.Membrane integral proteins (group A Nonpolar:(Methionine Met,M) (Phenylalanine Phe,F,) (Isoleucine Ile,I) (Tryptophan Trp W) (Leucine leu,L)(Alanine Ala,A) 2.Anchored to the membrane covalent bonding Concept of Gradient They are selective barriers they are solvent, osmotic they move from highest to lowest, water movement osmotic pressure Passive Transport:Passive diffusion Increase rate of glucose 1 Active transport: Na/K ATPase Movement of molecules against a gradient directly linked to hydrolysis of high-energy, exchange of Na to K,AT hydrolysis 2 Active Transport galactoside permease Transport of galactoside across bacteria Movement of molecules against a gradient linked to the movement of other molecules following their gradient , inside lower concentration and outside higher concentration Membrane Transport Passive transport driven by a concentration gradient simple diffusion :a molecule or ion moves through an opening facilitated diffusion: a molecule or ion is carried across a membrane by a carrier/channel protein Active transport: a substance is moved against a concentration gradient primary active transport :transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ion pump secondary active transport:driven by H+gradient Chapter Nine and Ten Nucleic Acids Structure &DNa replication 1°structure: the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code 2°structure: the three-dimensional conformation of the polynucleotide backbone(double helix DNA) 3°structure: supercoiling only Prokaryotes 4°structure: interaction between DNA and proteins *Histones-only in Eukaryotes no in Prokaryotes Nucleic Acids Biopolymer made of monomer units (nucleotide) containing: a base derived from purine or pyrimidine (nucleobases) a monosaccharide, either D-ribose or 2-deoxy-D-ribose phosphoric acid Pyrimidine/Purine Bases Pyrimidine :Cytosine(in DNA and RNA) Thymine(in DNA some RNA), Uracil (only in RNA) Purine:Adenine(DNA and RNA) Guanine (in DNA and RNA) Nucleosides:Compounds that consists of D-ribose or 2-deoxy-D-ribose covalently bonded to a nucleobase by a -N-glycosidic bond Example:Cytidine( A ribonucleoside in RNA )Deoxyguanosine ( A deoxyribonucleoside in DNA) 1.Nucleobase 2.Nucleotide (monosache nucleobase) 3. Nucleotide phosphate monosaccharides nucleophile RNA:Most of the time single stranded Transfer RNA:Transfer amino acids to site of protein synthesis(tRNA) Ribosomal RNA; Several Kinds variable in size combine with proteins(MRNA) To form ribosomes the site of protein synthesis Messenger RNA: Directs amino acids sequence of proteins(MRN) DNA 1 Strcuture Deoxyribonucleic acids, DNA:a biopolymer that consists of a backbone of alternating units of 2-deoxy-D -ribose and phosphate the 3’-OH of one 2-deoxy-D-ribose is joined to the 5’-OH of the next 2-deoxy-D-ribose by a phosphodiester bond Primary Structure:the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule base sequence is read from the 5’ end to the 3’ end System of notation single letter (A,G,C, and T) DNA-2°Structure Antiparallel 1.Nonpolar 2.Stocking effect of london forces Right handed helix ph 7 negative because of the phosphate Base Pairing Stiching effect weak dipole dipole of hydrogen bonds DNA-3°Structure Circular DNA:a type of double-stranded DNAin which the 5’ and 3’ ends of each strand are Joined by phosphodiester bonds. Supercoiling-Further coiling and twisting of DNA helix.(Topoisomerases, e.g. bacterial DNA gyrase)