BCHM 561 Final Flashcards
(61 cards)
Which is the strongest type of intermolecular attraction?
Covalent bonds
Which amino acid is most likely to be found on the surface of a protein
Glu
Which amino acid’s R group has a pKa that’s near physiological pH?
His
Which type of bond commonly stabilizes proteins that are secreted from the cell
Disulfide bonds
Efforts to produce short- versus long-acting synthetic insulin have focused on which of the following
altering the rate of hexamer dissociation
the pKa of the R group of aspartate is about 3.6. At what pH would there be 10 times as much of the protonated (HA) form relative to the unprotonated (A-) form
2.6
What is a dissociation constant (KD)? What does a high or low value indicate for a compound
Dissociation constants indicate the propensity for a compound to separate into two parts. A high KD indicates that a compound readily separates, while a low KD indicates that it tends to stay in one piece
what is cooperative binding? explain how it affects the affinity of hemoglobin for oxygen
cooperative binding is where binding of a multimeric protein to a ligand affects the binding affinity of its other subunits. Hemoglobin’s affinity for oxygen increases as more subunits are bound to oxygen
What is the isoelectric point (pI) of a protein? What technique separated proteins based on their pI
the isoelectric point is the pH at which the protein has a net neutral charge. Isoelectric focusing separates proteins based on the pI
How does an affinity chromatography column bind a specific protein? How do you elute your protein of interest from the column after it’s been bound?
an affinity chromatography column has beads coated with a ligand that your protein binds to and captures the protein as it flows through the column. You can elute your protein from the column by adding a solution containing a high concentration of the ligand
what is unique about prions as infectious agents that made disease experts skeptical of their existence at first? How do prions cause neurodegenerative disease once they get into a cell
prions are infectious proteins that were controversial because they transmit disease without involving DNA or RNA like viruses and cellular pathogens. Prions cause disease by binding to other non-prion proteins and causing them to adopt the prion fold
What does M/z mean on the x-axis of a MALDI-TOF graph? Why does it give us this value instead of the molecular mass?
M/z indicates the mass-to-charge ratio of the particles. We get M/z because both the mass and the charge affect how quickly a particle travels to the detector in a mass spectrometer
Which of the peaks in a MALDI-TOF graph likely corresponds to your protein’s molecular ion peak? Why?
The peak that is the largest and is closest to the predicted mass
What do the other peaks in a MALDI-TOF graph likely represent
the right-hand peak might be a dimer of our protein with one H+ because its M/z is about twice of the molecular peak. The left hand peak might be our protoxin with two H+ charges because its M/z is about half the molecular peak
What types of bonds must be broken before you can run mass spec on a protein of interest? How do we prevent these bonds from reforming after we break them?
Disulfide bonds must be broken. This is done by reducing and then chemically modifying the cysteine residues so they can’t re-form the disulfide bonds
Can a dipeptide rotate around a peptide bond?
No, because peptide bonds have partial double-bond character due to resonance
SDS-PAGE is a gel-based method of separating proteins. How does an SDS-PAGE gel separate proteins? What type of proteins travel down an SDS-PAGE gel the quickest and why is the pattern different from gel-filtration chromatography?
In SDS-PAGE, the gel has small pores that are easier for smaller proteins to fit through. Thus, smaller proteins move through the gel faster. Unlike gel-filtration chromatography, there aren’t any gaps for the larger proteins to travel through and thus they’re slowed down more than the smaller proteins
What does HPLC stand for? How would HPLC enhance the resolution of your gel-filtration experiment?
HPLC stands for high-performance liquid chromatography. HPLC uses liquid flow and much smaller beads with more interaction sites to give greater resolution between different proteins
What’s the difference between a primary and a secondary antibody in a western blot? Why don’t we use the primary antibody only?
primary antibodies recognize the antigen (protein) of interest, and secondary antibodies bind to the primary and are linked to something that generates the detectable signal. We don’t use only primary antibodies because it would be unfeasible to generate enzyme- or fluorophore-linked primary antibodies for every protein you would want to detect
Purification tables will record specific enzyme activity of a sample. What is specific activity> How is it different from total activity?
Specific activity is enzyme activity / amount of protein. Total activity is simply the total amount of enzyme activity in your whole sample without regard to amount of protein.
If you have two proteins you want to separate that are the same mass but have very different charges, what method could you use?
Ion-exchange chromatography
Consider the reaction A + B»_space; A-B. Could A or B be acting as an enzyme?
No
Which of the following is not a characteristic of an enzyme?
They lower the energy level of the substrate(s)
What is the order of the reaction A + B > C?
Second Order
