Term
| What are the 6 non polar amino acids? What does that mean for its inherent net charge?Draw them |
|
Definition
| glycine, alanine, valine, isoleucine, and methionine. the net charge on the amino acid is always zero |
|
|
Term
| What are the aromatic amino acids? What special capability to they have?Draw them |
|
Definition
| Phenylalanine, tyrosine, and tryptophan. They can fluoresce under Uv light. |
|
|
Term
| What are the 6 polar amino acids? Draw them |
|
Definition
| serine, proline, threonine, cysteine, asparagine, glutamine |
|
|
Term
| What are the positively charged amino acids? Draw them |
|
Definition
| lysine, histidine, arginine |
|
|
Term
| What are the negatively charged amino acids? Draw them |
|
Definition
|
|
Term
| What is the hybridization of water? Why? |
|
Definition
| The large electronegative difference between O and H causes a 33% ionic character and a large dipole |
|
|
Term
| What is the H bond energy? |
|
Definition
|
|
Term
| Draw the best overlap of H bond orbitals |
|
Definition
| a strong H bond is characterized by strong straight up and down H bond, whereas a weak bond is at an angle[image] |
|
|
Term
| What are some instances in which H bonding could occur? |
|
Definition
hydroxyl to a wataer
oxygen carbonyl to a water
complimentary bases of DNA |
|
|
Term
| how many H bonds can water make? |
|
Definition
|
|
Term
| What does it mean that water has a high dielectric constant? |
|
Definition
| the forces between two charges decrease, allowing solvation of ions |
|
|
Term
| does hexane have a larger or smaller dielectric constant than water? |
|
Definition
| smaller, causes force between charges to increase 30 to 40 times |
|
|
Term
|
Definition
| Just draw water surrounding it[image] |
|
|
Term
| What is an example of a substance that has a higher dielectric constant than water? |
|
Definition
|
|
Term
| Draw how micelles increase the entropy of water |
|
Definition
There isn't really an ordered shell of water molecules surrounding it[image]
releases maximum number of water molecules |
|
|
Term
| What does it mean when an ion is mobile? |
|
Definition
| The distance an ion moves in one second under the electric field of 1V/cm |
|
|
Term
| Why are acid - base reactions so fast? |
|
Definition
| H+ ion has very quick migration in water due to proton jumping |
|
|
Term
| What is the Henderson-Hasselbach equation? |
|
Definition
|
|
Term
| What are 3 good biological buffers? |
|
Definition
| phosphate, histidine, bicarbonate |
|
|
Term
| write out the derivation of the henderson hasselbach equation from [image] |
|
Definition
|
|
Term
| What is pH of solution of 0.1 M NaH2PO4 and 0.13 M Na2HPO4? ...pka is 7.2 |
|
Definition
|
|
Term
| What is pH of 1 L of 0.1 M H3PO4 and 150 ml of 1 M NaOH? pka is 7.2 |
|
Definition
|
|
Term
| What is pH of 1 L of 0.1 M NaH2PO4 and 130 ml of 1 M NaOH? |
|
Definition
|
|
Term
| What is pH of 0.1 M acetic acid? pK is 4.5 |
|
Definition
|
|
Term
| What is pH of 0.1 M NaOAc? |
|
Definition
|
|
Term
| What is the difference between delta G not and delta G not prime? |
|
Definition
| Go’ can be thought of as the “non standard” ΔGo where reactant concentrations are not 1 M |
|
|
Term
| In coupled reactions, are equilibrium constants multiplied or added? What about free energies? |
|
Definition
equilibrium constants: multiplied
free energy: additive |
|
|
Term
| What is the common way to couple an unfavorable reaction? |
|
Definition
|
|
Term
| What are the typical Pkas of the carboxyl and amino groups? |
|
Definition
|
|
Term
| What are the 3 letter abbreviations for the nonpolar amino acids ? Draw them. |
|
Definition
| Gly, Ala, Val, Leu, Ile, Met |
|
|
Term
| What are the 3 letter abbreviations for the aromatic amino acids? Draw them |
|
Definition
|
|
Term
| What are the three letter abbreviations for the polar amino acids? |
|
Definition
| Ser, Pro, Thr, Cys, Asn, Gln |
|
|
Term
| What are the three letter abbreviation for the positively charged amino acid groups? |
|
Definition
|
|
Term
| What are the 3 letter abbreviations of negatively charged amino acids? |
|
Definition
|
|
Term
| What are the one letter abbreviations of all the non polar amino acids? |
|
Definition
|
|
Term
| What are the one letter abbreviations of all the aromatic amino acids? |
|
Definition
|
|
Term
| What are the one letter abbreviations of all the polar amino acids? |
|
Definition
|
|
Term
| What are the one letter abbreviations of the positively charged amino acids? |
|
Definition
|
|
Term
| What are the one letter abbreviations of the negatively charged amino acids? |
|
Definition
|
|
Term
| how do you break a disulfide bond? how do you form one? |
|
Definition
to break: reduce it
to form: oxidize it. |
|
|
Term
| are disulfide bonds found in intracellular or extracellular proteins? |
|
Definition
| found in extraceullar proteins |
|
|
Term
| Why does it make sense that you won't find many disulfide bonds inside a cell? |
|
Definition
| it is a reducing environment inside the cell |
|
|
Term
| how do disulfide bonds provide structure to proteins? |
|
Definition
|
|
Term
| determine the pI of (0)Gly-(4)Glu-(6)His-(11)Lys-(0)Ala |
|
Definition
|
|
Term
| determine the net charge of (0)Gly-(4)Glu-(6)His-(11)Lys-(0)Ala under pH 6 and 7 |
|
Definition
|
|
Term
| which amino acid does not rotate polarized light? |
|
Definition
|
|
Term
| what is the main way to describe different enantiomers? |
|
Definition
| by the direction that they rotate the light. L-left, D-right |
|
|
Term
| What do D and L stand for? |
|
Definition
D- Dextrorotator
L - levorotatory |
|
|
Term
| All amino acids in proteins have which stereochemical rotation? L or D? |
|
Definition
|
|
Term
What are two non-standard amino acids found in collagen ?
Draw them. |
|
Definition
| 4-hydroxyproline and 5-hydroxylysine[image] |
|
|
Term
| What are post translational modifications that can be put on histone proteins? |
|
Definition
methylated, acetylated or
phosphorylated |
|
|
Term
| What is the N-terminal amino acid in prokaryotes, but then removed? Draw it. |
|
Definition
| N-formylmethionine[image] |
|
|
Term
| What is a nonstandard amino acid found in several proteins associated with blood clotting? Draw it |
|
Definition
|
|
Term
| Where can you find selenocysteine? Draw it. |
|
Definition
| sometimes you can find it at the UGA stop codon [image] |
|
|
Term
| Where can you find Pyrrolysine? Draw it. |
|
Definition
| found in proteins of methanogenic archaea used for producing methane and is coded with codon UAG (start )[image] |
|
|
Term
| What makes something prochiral? |
|
Definition
| If you replace one one substituent and the achiral center turns into a chiral center, it is prochiral |
|
|
Term
| draw the re and si faces of acetylaldehyde. Why are these signigifcant? |
|
Definition
| If you replace one subsistent with deuterium, then different enantiomers are produced depending on which side they were added to [image] |
|
|
Term
| Explain the significance of the chirality of life |
|
Definition
Ordinary synthesis of chiral molecules produces racemic mixtures. Ordinary methods do not show stereochemical preference.A fact of life: biosynthesis of substances having asymmetric centers almost produce pure stereoisomers.
• Using this criteria, examination of amino acids in meterorites always show racemic mixture. Thus not based on life. |
|
|
Term
| What is the most abundant substance in cells? |
|
Definition
|
|
Term
| What type of solution is good for lysing cells? |
|
Definition
|
|
Term
| What does lysozyme degrade? |
|
Definition
|
|
Term
| What does a french press do? |
|
Definition
| shears cells by putting them in high pressure and squirting them through small orifice |
|
|
Term
| How does a solicitor break open cells? |
|
Definition
| with high intensity sound waves |
|
|
Term
| How do proteases become an issue during cell lysis? |
|
Definition
| they can start degrading the proteins if the cell lysate isn't kept on ice with protease inhibitors |
|
|
Term
| What are five characteristics of proteins that can be leveraged to separate them ? |
|
Definition
| solubility, ionic charge, polarity, molecular size, binding specificity |
|
|
Term
| what are 2 purification techniques to separate proteins via solubility? |
|
Definition
|
|
Term
| what are 3 purification techniques to separate proteins via ionic charge? |
|
Definition
| ion exchange chromatography, electrophoresis, isoelectric focusing |
|
|
Term
| what are 4 purification techniques to separate proteins via polarity? |
|
Definition
| adsorption chromatography, paper chromatography, reverse phase chromatography, hydrophobic interaction chromatography |
|
|
Term
| what are 4 purification techniques to separate proteins via molecular size? |
|
Definition
| dialysis and ultrafiltration, gel electrophoresis, gel filtration chromatography, ultracentrifugation |
|
|
Term
| what is 1 purification technique to separate proteins via binding affinity? |
|
Definition
|
|
Term
| Explain how you would use techniques of salting in and out to purify proteins in a mixture. |
|
Definition
| Add in the maximum amount of salt that the protein can stand to be pulled down by. Centrifuge and Discard supernatant. Add the smallest amount (concentration) of salt that can pull down the protein. pellet is protein |
|
|
Term
| When are proteins least soluble? Why is this? |
|
Definition
| When net charge of protein is zero, this is the isoelectric point or pI. Proteins are typically least soluble at their pI due to minimizing charge charge interactions. |
|
|
Term
| How would you crystallize a protein? |
|
Definition
| bring protein solution past its saturation point with different types of precipitating agents. Over time the protein will fall out of solution |
|
|
Term
| Describe an ELISA step by step: |
|
Definition
1. immobilize first antibody on solid support
2. incubate with protein-containing sample
3. add a second antibody that is covalently linked to an assayable enzyme
4. wash an assay the enzyme |
|
|
Term
| What is an ELISA used for? |
|
Definition
| to detect small amounts of specific proteins and other biological substances in both laboratory and clinical applications. |
|
|
Term
| What does ELISA stand for? |
|
Definition
| enzyme-linked immunosorbent assay |
|
|
Term
| What two properties of proteins do chromatographic methods leverage? |
|
Definition
|
|
Term
| What is the charge of the protein if the pH is above the pI? |
|
Definition
|
|
Term
| What is the charge of the protein if the pH is below the pI? |
|
Definition
|
|
Term
| What is the charge of an anion exchange resin? |
|
Definition
|
|
Term
| Write a chemical reaction that represents how anion exchange chromatography works |
|
Definition
|
|
Term
| If you wanted to elute a negatively charged protein, what kind of resin would you use, and what pH should you use relative to the protein's pI? |
|
Definition
| use a resin with a positive charge, like DEAE cellulose or sephadex. use a pH above the pI of the protein. Protein of interest adheres and drive off with salt gradient. |
|
|
Term
| using anion exchange chromatography, (positively charged resin), do proteins with the highest or the lowest pIs elute first? |
|
Definition
| Proteins with highest pIs elute first |
|
|
Term
| Which resin would you use if you wanted to isolate a positively charged protein? |
|
Definition
| CM-cellulose or Sephadex. (negatively charged resin) |
|
|
Term
| If you are using cationic exchange, what pH should you use relative to the pI ? |
|
Definition
|
|
Term
| in cationic exchange, do proteins with high pIs or low pIs elute first? |
|
Definition
|
|
Term
| What does DEAE stand for in DEAE cellulose? is it basic or acidic? |
|
Definition
| diethylaminoethyl - weakly basic |
|
|
Term
| what does CM - in CM-cellulose stand for? Is it basic or acidic? |
|
Definition
| carboxymethyl - weakly acidic |
|
|
Term
| would DEAE separate acidic or basic proteins ? |
|
Definition
|
|
Term
| does CM- cellulose separate acidic or basic proteins? |
|
Definition
|
|
Term
| What does gel filtration separate proteins by? |
|
Definition
|
|
Term
| What is another name for gel filtration chromatography? |
|
Definition
| molecular sieve chromatography or size exclusion |
|
|
Term
| What is an exclusion limit? |
|
Definition
| in gel filtration, the gel's exclusion limit is the molecular mass of the smallest molecule unable to penetrate the pores of a given gel |
|
|
Term
| through what unit are proteins measured by the order they come through a gel filtration ? |
|
Definition
| relative elution volume or V(e)/V(0) |
|
|
Term
| What is V(t), V(x), V(0) and V(e)? |
|
Definition
V(t) = total bed volume of the column
V(x) = volume occupied by the beads
V(0) = volume of space around the beads
V(e) = elution volume, or the amount of solvent needed to elute the solute
**in gel filtration chromatography |
|
|
Term
| Which types of proteins will elute first in gel filtration? |
|
Definition
| those with the largest molecular masses |
|
|
Term
| What are examples of what gels can be made out of? |
|
Definition
| polyacrylamide, agarose, dextrose |
|
|
Term
| What does a dialysis separate proteins based on ? |
|
Definition
| size --- smallest proteins elute first |
|
|
Term
| What is the size cutoff for a dialysis bag? |
|
Definition
|
|
Term
| How does affinity chromatography work? |
|
Definition
| the column has ligands specifically for the protein of interest is covalently attached to a porous membrane. The protein of interest binds to the ligand and the others pass through the membrane. Then the protein is eluted off the membrane and collected. |
|
|
Term
| What is the mechanism for cross linking a gel? Draw it out! |
|
Definition
|
|
Term
| How was the insulin receptor purified? |
|
Definition
|
|
Term
| what if a protein needs to be purified by affinity chromatography but doesn't have a known ligand? |
|
Definition
| GST - ( glutathione S - transferase) is attached at the N terminus and run through a glutathione agarose column |
|
|
Term
| How do you calculate stock activity? What are the units? |
|
Definition
| (absorbance/extinction coefficient) * (10^6umol/10^3mL), then divide by "amount added" ...units are UNITS/mL |
|
|
Term
| How do you calculate specific activity? What are the units? |
|
Definition
| stock activity (units/mL) / stock concentration (mg/mL) = specific activity = units/mg |
|
|
Term
| How do you calculate total activity ? What are the units? |
|
Definition
| stock activity (units/mL) * total volume (mL) = total activity (units) |
|
|
Term
| How do you calculate the % recovery ? units? |
|
Definition
| total activity of the new / total activity of the crude * 100 |
|
|
Term
| How do you calculate fold purification? |
|
Definition
| new specific activity / specific activity crude = fold ( unitless) |
|
|
Term
| What does paper electrophoresis separate proteins based on? |
|
Definition
| ionic charge. The dot applied initially to the cellulose acetate paper will migrate towards the cathode or the anode based on its charge. |
|
|
Term
| What is the difference between paper electrophoresis and paper chromatography? |
|
Definition
| paper electrophoresis separates based on ionic charge, whereas paper chromatography separates based on polarities |
|
|
Term
| How is the polymerization of acrylamide and N,N Methylenebiacrylamide initiated ? |
|
Definition
| induced by free radicals from the chemical decomposition of ammonium persiflage or the photodecomposition of riboflavin in traces of O2. |
|
|
Term
|
Definition
|
|
Term
| What does SDS-PAGE stand for? What does it do? |
|
Definition
| sodium dodecyl sulfate polyacrylamide gel electrophoresis...separates based on size...can determine purity and molecular weight |
|
|
Term
| Detail the steps of a Western blot: |
|
Definition
1. transfer (blotting a gel onto nitrocellulose)
2. block with casein
3. wash // stain with anti-protein (antibody 1)
4. wash// stain with secondary (enzyme linked)
5. assay linked enzyme |
|
|
Term
| What does SDS do to proteins and how? |
|
Definition
| SDS imparts masks the protein's intrinsic charge so that SDS-treated proteins tend to have the identical charge-to-mass rations and similar shapes |
|
|
Term
| What is the goal of isoelectric focusing? (IEF) |
|
Definition
| separation by pI.....If a mixture of proteins is electrophoresed through a solution having a stable pH gradient, in which the pH smoothly increased from anode to cathode, each protein will migrate to the position on the gel corresponding to its pI |
|
|
Term
| How are 2-D electrophoresis and isoelectric focusing related? |
|
Definition
| 2-D techniques allow the proteins to be separated on the x axis by pH and separated on the y axis by molecular weight |
|
|
Term
|
Definition
| aggregate of all proteins in a cell or organism |
|
|
Term
| Why would you want to use MALDI/TOF? |
|
Definition
| allows for identification of proteins via peptide mapping |
|
|
Term
| How would you do MALDI/TOF? |
|
Definition
| 2-D technique. Isolate protein, treat with trypsin, extract peptide fragments, analyze via MALDI/TOF, identify protein via the peptide fragmentation and protease used...complete ID of extracellular proteins with Mass Spec |
|
|
Term
|
Definition
| cleaves on the C-side of Arg or Lys |
|
|
Term
| chymotrypsin cleaves where? |
|
Definition
| cleaves on the C-side of Phe, Tyr, Trp |
|
|
Term
| Cyanogen bromide cleaves where? |
|
Definition
|
|
Term
| Dansyl chloride does what? |
|
Definition
| reacts with the N-terminus, does acid hydrolysis to break peptide bonds, detected by HPLC |
|
|
Term
|
Definition
|
|
Term
| What does Edman's reagent do? |
|
Definition
| removes one amino acid at a time, can be used for sequencing proteins |
|
|
Term
| how many amino acids can Edman's reagent remove consistently? |
|
Definition
| 20-30 from the N-terminus |
|
|
Term
| If Edman's reagent cleaves from the amino terminus, what enzyme cleaves from the carboxyl terminus? |
|
Definition
|
|
Term
| When using carboxypeptidase, what do we know about the first amino acid that comes off? |
|
Definition
| it's the one with the highest concentration |
|
|
Term
| Name the exopeptidase that cleaves from the C-terminus |
|
Definition
|
|
Term
| Can carboxypeptidase be used for sequencing? |
|
Definition
| no---it has different specificity ...for example if the second amino acid cleaved faster than the first, then you would see the first two together. and it is not active on Pro residues |
|
|
Term
| what is hydrazinolysis used for? |
|
Definition
| used for sequencing and determining the C-terminal amino acid |
|
|
Term
| What are the steps from going from a disulfide bond to acetylated cysteine residues |
|
Definition
| reduced by dithioteitol, acetylated by iodoacetate |
|
|
Term
| How does a disulfide bond form two separate cysteic acid residues? |
|
Definition
| oxidation via perfomic acid |
|
|
Term
| Name 5 endopeptidases and where they cleave |
|
Definition
trypsin, chymotrypsin, elastase, thermolysin, pepsin
ALL CLEAVE AT SCISSLE PEPTIDE BOND (between c=o and NH group) |
|
|
Term
| How does cyanogen bromide cleave peptides? |
|
Definition
| the lone pair on sulfur displaces Br and then O=C lone pair pushes complex off. [image] |
|
|
