ACS BIOCHEMISTRY EXAM – OBU Latest Graded A+

This study source was downloaded by from CourseH on :12:27 GMT -06:00 ACS BIOCHEMISTRY EXAM – OBU Latest Graded A+ Separation of Organelles / Fractionation of Cells - ANSWER Differential Centrifugation 1. Tissue Homogenization 2. Low speed centrifugation (whole cells, nuclei, membrane) 3. Medium speed centrifugation (mitochondria, lysosomes, peroxisomes) 4. High speed centrifugation (microsomes -fragments of ER) 5. Very high-speed centrifugation (soluble proteins) Isopycnic Centrifugation - ANSWER Sucrose-Density (components separated by density) Interactions occur... - ANSWER because of the electrostatic properties of molecules. - these properties can involve full, partial or momentary charges Examples of electrostatic interactions - ANSWER dipole-dipole interactions hydrogen bonds ionic interactions van der Waals interactions Hydrophobic Interactions... - ANSWER entropy, not an attraction between hydrophobic molecules The special properties of water... - ANSWER derived from its polarity and hydrogen bonding - overall polarity is due to polar bonds and shape Entropy & 2 Critical Processes that involve Water - ANSWER 1. Dissolving of highly ordered solids (salts) - entropy of some of the water is decreased, but the entropy of the ions is greatly increased 2. The hydrophobic effect - there is no strong attraction between hydrophobic molecules (van der Waal's only) - there is no repulsion between hydrophobic molecules and water Aggregation of H-bonds in water is driven by - ANSWER entropy change of water (decrease) - it can also drive other reactions such as enzyme-substrate interaction This study source was downloaded by from CourseH on :12:27 GMT -06:00 Ion Product Constant - ANSWER [H+][OH-] = Keq [HOH] = 1.0 x 10^-14 This study source was downloaded by from CourseH on :12:27 GMT -06:00 Equilibrium Constant - ANSWER Keq = [H+][OH-]/[HOH] 2 Applications of pH - ANSWER 1. Controlling pH with buffers 2. How pH (protonation) affects weak interactions of molecules (charge and ability to form H-bonds) pKa = - ANSWER -log Ka pKa definition - ANSWER pH at which half of the acid is ionized. titration curves reveal the pKa of weak acids - is a measure of affinity for protons a high pKa constitutes... - ANSWER it takes a high pH (lots of OH-) to remove the H+ from the acid - the acid has a high affinity for H+ - if the pKa is high, it has a HIGH affinity (want) to grab onto H+, because of the large amount of OH- Significance of Buffering - ANSWER 1. Homeostasis 2. This phenomenon is very important for biochemical activity - many reactions depend on the state of protonation of particular functional groups A buffer is made... - ANSWER from a weak acid and its conjugate base Henderson-Hasselbalch Equation - ANSWER pH = pKa + log ([A-]/[HA]) (unprotonated over protonated) Decent Buffering occurs... - ANSWER within around 1 pH unit of pKa (ration of A/HA is between 1/10 or 10/1 Buffering of Blood - ANSWER blood is primarily buffered by the bicarbonate system (CO2/HCO3-) but also by phosphoric acid system (H2PO4-/H3PO4) The hydration of CO2 (middle equilibrium) is greatly enhanced by the enzyme... - ANSWER carbonic anhydrase Carbonic anhydrase - ANSWER raises H+ conc in RBC's, therefore affecting the ionization of hemoglobin side chains which affects O2 binding and release How will breathing rate affect the CO2/HCO3- equilibrium? - ANSWER excessive breathing creates a low level of CO2 in the blood - if CO2 levels increase, the brain tells the lungs to increase rate and depth of breathing to bring the levels down This study source was downloaded by from CourseH on :12:27 GMT -06:00 Shallow or slow breathing... - ANSWER will eventually cause elevated blood CO2 levels, equilibrium to shift to the right, the H3O+ conc. increases and pH drops Inside cells buffering is achieved by... - ANSWER phosphate ions and amino acid side chains on proteins Non-polar, aliphatic R groups - ANSWER glycine, alanine, proline, valine, leucine, isoleucine, methionine Aromatic R groups - ANSWER phenylalanine, tryptophan, tyrosine Polar, uncharged R groups - ANSWER serine, threonine, cysteine, asparagine, glutamine Negatively charged R groups - ANSWER aspartate, glutamate Positively charged R groups - ANSWER lysine, arginine, histidine PI (isoelectric point) - ANSWER the pH value at which the molecule carries no electrical charge. Which atoms around the peptide bond are coplanar? - ANSWER O C-C-N-C H Column Chromatography - ANSWER Amino Acids and proteins can be separated based on their interactions with a stationary phase packed into a column 1. Don't stick > they come off with the effluent 2. Stick to the column or interact with the matrix > their flow is slowed. Derivative and HPLC chromatography - ANSWER Mass Spec - ANSWER Size Exclusion (Gel filtration chromatography) - ANSWER - protein mixture is added to column containing cross-linked polymer - protein molecules separate by size; larger molecules pass more freely appearing in the earlier fractions Affinity chromatography - ANSWER -solution of ligand is added to column - protein mixture is added to column containing a polymer-bound ligand specific for protein of interest -unwanted proteins are washed through the column - protein of interest is eluted by ligand solution Electrophoresis - ANSWER - cross linked polymers act as molecular sieves This study source was downloaded by from CourseH on :12:27 GMT -06:00 (A. polyacrylamide B. agarose) - an electric field moves the proteins thru the matrix migration is determined by charge, and size and shape of the molecule 3 Types of Electrophoresis - ANSWER 1. Native electrophoresis 2. SDS electrophoresis (sodium-dodecyl sulfate) 3. Isoelectric focusing 2-D gels - ANSWER 2-dimensional electrophoresis combines isoelectric focusing and SDS gel Isoelectric focusing - ANSWER - establish a pH gradient using an electric field and electrolytes in a gel tube or plastic strip - apply protein sample to Gel > proteins migrate through the pH gradient until they reach their isoelectric point - after staining, proteins are shown to be distributed along the pH gradient according to their pI values. How do we get a pure sample of a protein to work with? - ANSWER protein purification Steps of Protein purification - ANSWER 1. Break open the cells that have protein of interest (osmotic lysis) 2. Protein fractionation (precipitation, column chromatography) Standard Sequencing Method for Proteins - ANSWER 1. Determine AA composition, identify amino terminal using FDNB, reduce and alkylate cysteine residues 2. Fragment into peptides of sequence-able length (enzymatic digestion with trypsin/chymotrypsin, chemical cleavage with cyanogen bromide) 3. Separate and purify the peptides (gel electrophoresis, liquid chromatography) 4. For chemical sequencing, adsorb the peptides onto a solid support Secondary Structure - ANSWER small scale, local conformation of a part of a peptide (alpha helix and beta sheets) Why determine protein sequence? - ANSWER AA sequence > 3-D structure > Function Tertiary Structure - ANSWER overall 3D structure of an entire polypeptide Quaternary Structure - ANSWER arrangement of protein chains (subunits) into complexes Chemical Synthesis of Peptides - ANSWER Merrifield Synthesis - blocking reactive groups on AA's to control which reactive group participates in a given reaction. - COOH groups are made more reactive by using DCC This study source was downloaded by from CourseH on :12:27 GMT -06:00 Enzyme activity - ANSWER refers to the total units of enzyme in a solution Specific activity - ANSWER is the number of enzyme units per milligram of total protein - it is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure. Polarity in the peptide - ANSWER the peptide BOND exhibits resonance and polarity Alpha Helix stabilizing force - ANSWER the internal H-bonds between carboxyl O's and amide N's along the helix 5.4 angstroms per 1 turn of the helix - ANSWER 3.6 AA per turn Unfavorable Helix AA - ANSWER pro, phe, ile, cys, asn, ser, thr, gly (too flexible) (-)(acidic) AA's are often found at the - ANSWER N-terminus of helices (+)(basic) AA's are often found at the - ANSWER C-terminus of helices Acidic AA's ... - ANSWER lose their proton at low pH Asp and Glu have side chains with carboxylic acids whose pKa's are low enough... - ANSWER to lose protons, therefore becoming negatively charged/acidic amino acids Beta-sheets - ANSWER extended zig zag conformation with multiple strands beside each other in a sheet *antiparallel sheets have close to optimal H-bond angles (parallel have less optimal) Ramachandron Plot - ANSWER plot the different angle possibilities that the peptide bonds can assume - only a few combinations are in stable secondary structures Contiguous segments of a protein that are hydrophobic can be identified using a... - ANSWER hydropathy plot hydropathy plot - ANSWER used to predict the number and position of transmembrane segments in a given protein sequence Motif - ANSWER secondary structures can fold into characteristic patterns Domain - ANSWER a structure thats stable apart from the rest of the protein Tertiary structures are held together by - ANSWER hydrophobic effect covalent linkages salt bridges This study source was downloaded by from CourseH on :12:27 GMT -06:00 combination of weak interactions 2 Major tertiary structures - ANSWER 1. Fibrous (sheets or long strands) 2. Globular (much more diverse) Alpha keratin - ANSWER right handed helices that form one left handed super helix Collagen - ANSWER 3 left handed helices twisted into a right handed super helix Much of the stability of the folded protein is due to... - ANSWER maximization of entropy AA sequence determines... - ANSWER tertiary structure All necessary info for proteins to fold is found in... - ANSWER the amino acid sequence Proteins fold while being synthesized, they tend to start at... - ANSWER the amino terminus, rather than all at once Some proteins need help (re)folding. can use these - ANSWER molecular chaperones Hsp70 proteins chaperonins Heme + Iron = - ANSWER a prosthetic group Hemoglobin binds O2 ... - ANSWER cooperatively Allosteric protein: the binding of a ligand at one site affects binding at other sites - ANSWER the binding of a ligand at one site affects binding at other sites Induced fit: - ANSWER binding of ligand changes protein conformation and energy of binding Sigmoid curve - ANSWER diagnostic for cooperative binding Hill coefficient greater than 1 - ANSWER positive cooperativity Hill coefficient less than 1 - ANSWER negative cooperativity H+ affects the cooperativity of O2 binding to Hb... - ANSWER by affecting the protonation of His HC3 (stabilizing T-state) When blood gets more acidic in the tissues... - ANSWER more HisHC3 side chains are protonated, which makes a salt bridge that stabilizes the T-state and the release of O2. This study source was downloaded by from CourseH on :12:27 GMT -06:00 BPG regulates O2 binding - ANSWER interacts with (+) side chains around a hole in the middle of the deoxy Hb tetramer. Stabilizes T-state, reduces affinity for O2 At high altitudes tissues have less O2... - ANSWER more BPG is formed and Hb has reduced affinity in the tissues and releases more O2 antibodies or immunoglobulins bind... - ANSWER antigens Immunoglobulin fold = - ANSWER stack of 2 Beta sheets Antigen binding region of each chain is a combination of... - ANSWER the variable regions and heavy and light chains Interaction with antigen causes a change in conformation of the protein = - ANSWER induced fit Myosin (2 heavy chains and 4 light chains) - ANSWER moves along filaments of actin, which attach to Z disks When ATP binds... - ANSWER reduces affinity for actin