BICH 410 1st Edition Exam 2 Study Guide Lectures 1 12 Protein Techniques What can denature proteins Heat pH detergents sds chaotropic agents urea guanidium chloride What did chirstian ansinsin discover He realized that ribonuclease A could be denatured and could then spontaneously refold based solely on the information in the amino acid sequence What are the steps required for protein purification Extract protein from cell ie solubilization fractionation to separate desired protein from the rest of the proteins and visualize ie colormetric or UV to confirm protein purity and identity How is purity determined Amount of desired protein total protein purity For enzymes specific activity total activity purity What characteristics can be used for protein separation SOLUBILITY pH proteins least soluble at pI salting in out ionic charge ion exchange chromatography electrophoresis isoelectric focusing polarity hydrophobic interaction chromatography size gel filtration chromatography ultracentrifugation and SDS PAGE binding specificity affinity chromatography What is the difference between salting in and out When adding salts the protein protein interactions are inhibited and increase solubility but when too much salt is added the salt interferes with the protein water interaction causing precipitation When using gel filtration which molecules elute first The largest molecules come out first while the smallest come out last because they have to travel through more pores in the column since the molecules can fit into more pores Explain how an anionic exchange column works The proteins the bind to the column are anionic negatively charged consequently the anionic column is actually cationic The anionic proteins stick to the column while any cationic proteins flow through What does SDS do It allows subunit interactions to be eliminated as well as coating all molecules in negative charge allowing SDS Gel electrophoresis based solely on size What does isoelectric focusing do 1D Determines pI with pH gradient gel 2D separates by pH and by MW What are the steps for protein sequencing Separate subunits pH urea guanidine HCl of high salt conc break disulfide bonds reduce with 2mercaptoethanol or DTT and acetylate with iodoacetate or oxidize with performic acid end group analysis N terminus dansyl chloride FDNB phenylisothipcynate C terminus carboxypeptidase and exopeptidase cut protein and construct fragments Trypsin Arg Lys Chymotrypsin aromatic clostripain Arg cyanogen bromide met elastase G A pepsincleavage of aromatics at Nside and sequence each fragment edman s degradationphenylistothicynate repeat reconstruct sequence Difference between orthologs and paralogs Orthologs are from different species with homologous seq while paralogs are with same species Proteins with related function often have related sequence structure Myoglobin and Hemoglobin What are the major differences between Mb and Hb Mb is a monomeric protein that transports oxygen from tissue to tissue and its saturation curve is hyperbolic Mb also has a lower p50 than Hb because it muse bind tighter to O2 than Hb which must release O2 to the tissue Hb is tetrameric composed of a dimer ab of dimers a1b1 a2b2 dimer bound by hydrophobic ionic and hydrogen bonds while dimer of dimers does not use hydrophobic interactions therefore a change in pH will just effect dimer of dimers and transports O2 from lungs to tissue and its saturation curve is sigmoidal How does Oxygen bind and what colors can it cause the Fe to be The O2 binds to the heme FeII group which is corrdinated by 4 porphryin N atoms O2 solubility increased when bound to a carrier Oxygenated arterial FeII is red while deoxygenate veionous FeII is purple oxidized FeII FeIII causes a brown color aka methemoglobin CO binds 20 000 times tighter to an unbound heme then O2 but just 200x tighter with bound heme why is this This is due to steric interference This is because CO binds vertically which causes interference with distal His This does not occur with O2 because it arranges at an angle What is Kd and how is it calculated Kd is the dissociation constant opposite of Ka association constant 1 Kd the lower the Kd the higher the affinity MbO2 Mb O2 Kd Mb pO2 MB02 What is percent oxygen saturation Y and p50 Y of binding sites occupied total binding sites pO2 kd pO2 P50 is the affinity constant and a high p50 indicates low affinity and is the pO2 when 50 of binding sites are saturated with O2 Kd po2 at P50 Therefore binding is weaker when p50 is larger takes more pO2 TO REACH HALF SATURATION What is T vs R state T tense deoxyHb Fe is in heme plane R relaxed oxyHb Fe is out of heme plane What role does cooperativity play in Hb and how is it graphed As O3 binds and causes conformation change the adjacent subunits have increased affinity for O2 causing positive cooperativity N 1 on hillplot causing sigmoidal curve With cooperativity oxygen dissociation eqn po2n p50n po2n n 1 negative cooperativity n 1 no cooperative binding ie Mb hyperbolic Graphed with a Hill plot log y 1 y vs log po2 n slope of line wgeb y 5 and therefore log y 1y 0 and logp02 logp50 Graph will have 3 lines 1st 02 w lowest affinity 2nd and 3rd 02 with the t to r transition and 4 th O2 with hightest affinity Cooperativity allows for the release of O2 at low pressures ie tissues What allosteric interactions occur with Hb There can be homotropic same ligand and effector ie O2 of hetertropic and cause a conformational change either inducing more active R or less active T state This caused the Bohr effect where binding of H binds competitively with O2 the effect is caused by pka and ionic interactions With protonated Hb ionic interactions are stabilized preventing dissociation and increasing pKa but with O2 binding ionic interactions disrupted returning pKa THEREFORE low ph favors T stateor high p50 aka O2 low affinity Co2 also allosteric CO2 H20 HCO3 H causes decrease in pH again decreasing O2 binding CO2 can also transport CO2 carbamation which stabilizes ionic interactions favoring T state o Carbonic anhydrase accelerates CO2 rxn causing o2 depletion and msucles to make lactic acid BPG binds allosterically too allows Hb to become more oxygenated by binding to deoxy sites o Ie high altitude low PO2 increase BPG and increase in O2 to tissues occurs What abnormal Hb exist Fetal Hb which has higher affinity for O2 bc of different subunit composition hydroxyurea can cause HbF Oxidized FeIII causes cyanosis and