Biochem exam 1 - question Flashcards
E. coli is known as a gram-negative bacterial species. (a) How is this determined? (b) How do
gram-negative bacteria differ structurally from gram-positive bacteria?
: (a) Gram-negative bacteria have little affinity for the dye gentian violet used in Gram’s stain, but grampositive bacteria retain Gram’s stain. (b) Gram-negative bacteria have an outer membrane and a peptidoglycan
layer; gram-positive bacteria lack an outer membrane and the peptidoglycan layer is much thicker
Define a chiral center
A chiral center is a carbon atom that has four different substituents attached, and cannot be
superimposed on its mirror image – as a right hand cannot fit into a left glove
Differentiate between configuration and conformation
Configuration denotes the spatial arrangement of the atoms of a molecule that is conferred by the
presence of either double bonds, around which there is no freedom of rotation, or chiral centers, which give
rise to stereoisomers. Configurational isomers can only be interconverted by temporarily breaking covalent
bonds. Conformation refers to the spatial arrangement of substituent groups that, without breaking any bonds,
are free to assume different positions in space because of the freedom of bond rotation.
Give examples of 5 types of isomers.
See HM1
Explain why living organisms are able to produce particular chiral forms of different
biomolecules while laboratory chemical synthesis usually produces a racemic mixture. Is using a
racemic good? If one wants to use only one enantiomer, how would he/she do it?
Laboratory syntheses usually use achiral reagents and thus produce racemic mixtures of products. In
contrast, because all enzymes are made of chiral precursors, all enzymes are inherently chiral catalysts. Thus,
they will show strong stereoselectivity in reactants and mechanisms, leading to the production of chiral
products
Using a racemic is not good as biomolecules such as as receptors for drugs are stereospecific, so each of the
two enantiomers of the drug may have very different effects on an organism. One may be beneficial, the other
toxic; or one enantiomer may be ineffective and its presence could reduce the efficacy of the other enantiomer
Describe the principle of hydrophobic interactions chromatography; now describe the principle of
hydrophilic interactions chromatography
Hydrophobic Interaction Chromatography: Sample molecules containing hydrophobic and hydrophilic
regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of
sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the
media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a
decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity.
Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution
buffer
In hydrophilic interaction chromatography, is a liquid chromatography technique that uses a polar
stationary phase in conjunction with a mobile phase containing an appreciable quantity of water combined
with a higher proportion of a less polar solvent. Here, the hydrophilic, polar, and charged compounds are
retained preferentially compared with hydrophobic neutral compounds
Describe the principle of reversed phase chromatography; now describe the principle of normal
phase chromatography
In reversed phase chromatography, the most polar compounds elute first with the most nonpolar
compounds eluting last. The mobile phase is generally a binary mixture of water and a miscible polar
organic solvent like methanol, acetonitrile. Retention increases as the amount of the polar solvent (water)
in the mobile phase increases.
In normal-phase chromatography, the least polar compounds elute first and the most polar compounds
elute last. The mobile phase consists of a nonpolar solvent such as hexane or heptane mixed with a
slightly more polar solvent such as isopropanol, ethyl acetate or chloroform. Retention decreases as the
amount of polar solvent in the mobile phase increases.
Give the general Henderson-Hasselbalch equation and sketch the plot it describes (pH against
amount of NaOH added to a weak acid). On your curve label the pKa for the weak acid, and
indicate the region in which the buffering capacity of the system is greatest.
: The inflection point, which occurs when the weak acid has been exactly one-half titrated with NaOH,
occurs at a pH equal to the pKa of the weak acid. The region of greatest buffering capacity (where the titration
curve is flattest) occurs at pH values of pKa ±1. (See Fig. 2-17, p. 59.)
Briefly define “isotonic,” “hypotonic,” and “hypertonic” solutions. (b) Describe what happens
when a cell is placed in each of these types of solutions.
: (a) An isotonic solution has the same osmolarity as the solution to which it is being compared. A
hypotonic solution has a lower osmolarity than the solution to which it is being compared. A hypertonic
solution has a higher osmolarity than the solution to which it is being compared. (b) Higher osmolarity results
in osmotic pressure, which generally leads to movement of water across a membrane. In an isotonic solution,
in which the osmolarity of the solution is the same as the cell cytoplasm, there will be no net water movement.
In a hypotonic solution, water will move into the cell, causing the cell to swell and possibly burst. In a
hypertonic solution, water will move out of the cell and it will shrink.
Draw the structures of the amino acids tyrosine and aspartate in the ionization state you would
expect at pH 4.0.
low pH = COOH
high pH =NH2
neutral = zwitterion
See HM1
How does the shape of a titration curve confirm the fact that the pH region of greatest buffering
power for an amino acid solution is around its pK’s?
: In a certain range around the pKa’s of an amino acid, the titration curve levels off. This indicates that
for a solution with pH pK, any given addition of base or acid equivalents will result in the smallest change in
pH—which is the definition of a buffer.
Leucine has two dissociable protons, one with a pKa of 2.3, the other with a pKa of 9.7. Sketch a properly labeled titration curve for leucine titrated with NaOH; indicate where the pH = pK and the region(s) in which buffering occurs.
See the titration curve for glycine in Fig. 3-10, p. 79
Draw the structure of Lys–Ser–Glu in the ionic form that predominates at pH 4.
See HW1
low pH = COOH
high pH =NH2
neutral = zwitterionHW1
Draw the structure of a) Arginine, b) Aspartate, 3) Tyrosine and 4) +H3N-Arg-Asp-Tyr-COO- in
zwiterionic form and their ionized forms at low pH, neutral pH and high pH
See HW1 High pH = Everything is deprotonated. Low pH = Everything is protonated low pH = COOH high pH =NH2 neutral = zwitterion
Why do smaller molecules elute after large molecules when a mixture of proteins is passed
through a size-exclusion (gel filtration) column?
The column matrix is composed of cross-linked polymers with pores of selected sizes. Smaller
molecules can enter pores in the polymer beads from which larger molecules would be excluded. Smaller
molecules therefore have a larger three-dimensional space in which to diffuse, making their path through the
column longer. Larger molecules migrate faster because they pass directly through the column, unhindered by
the bead pores.
