EXAM 1 STUDY GUIDE
Chapter 2:
Noncovalent interactions: hydrogen bonds, electrostatic bonds, Van der Waals interactions
● O–H bond = 470 kJ/mol; O---H bond = 23 kJ/mol
● Noncovalent interactions are relatively weak. They are reversible at body temperature,
making them dynamic. The sum of many of these interactions is strong, as they hold
together proteins, DNA, RNA, and lipids.
● H-bond forms between covalently bonded O-H or N-H (electronegative atoms) and
adjacent H in a similar bond. H-bonds are strengthened by direction (linear H-bond is
stronger than an angled bond).
● Electrostatic bonds/salt bridges/ionic interactions: hydration shells block/mask charges
b/t ions, resulting in a decreased force…
● Van der Waals interactions result from transient dipole-induced dipole interactions of
electron clouds. ~4 kJ/mol. Tokay gecko has thousands of fine hairs that stick to walls via
Van der Waals.
Be able to do calculations with pH, Henderson-Hasselbalch equation; buffers.
● In biochemistry, weak acids and bases are critical. Weak acids and bases partly dissociate.
HA <--> H+ + A-.
● A buffer is a weak acid or base that resists the pH change of a solution. Works best when
[HA] = [A-]. There is usually a window for buffer area of +/- 1 pH.
● Henderson-Hasselbalch: pH = pKa + log{[A-]/[HA]}
● Physiological buffers include phosphate (intracellular buffer) and bicarbonate
(extracellular buffer)
Chapter 3:
Be able to draw the structures of the 20 amino acids at pH 7.0; know the three-letter codes and
one-letter codes for the amino acids. Know which ones are acidic, basic, hydrophilic, and
hydrophobic. L stereoisomers.
Be able to use the Henderson-Hasselbalch equation in calculations on amino acids.
Structure of the peptide bond
● Such a bond is formed by the removal of water
(dehydration) from the α-carboxyl group of one
amino acid and the α-amino group of another.
Peptide nomenclature, N- and C-terminal; disulfide bond
, Purification of proteins:
Column chromatography:
● Ion-exchange: separation by net electric charge of proteins at a given pH.
o Cation-exchange chromatography – solid matrix has negative charge.
o Anion-exchange chromatography – solid matrix has positive charge.
● Size-exclusion – Gel filtration. A molecular sieve of porous polymer beads separates
protein molecules according to their size. The beads have pores and cavities of a
particular size. Larger proteins cannot enter these cavities and take a shorter route path
thru the column.
Affinity chromatography (His-tag and Ni NTA column);
● Based on binding affinity. The beads in the column have a covalently attached chemical
group called a ligand – a group of molecule that binds to a macromolecule such as a
protein. This one-step separation process binds proteins with affinity to the ligand and
retards their movement. After the proteins that do not bind are washed thru the column,
the bound protein is eluted by a solution containing a high conc. of salt or free ligand.
● His-tag (engineered protein w/ a histidine tag)
● Nickel nitrilotriacetate (NiNTA). Nickel 2+ can bind proteins w/ multi-histidine tag. Wash
protein w/ histidine or imidazole containing buffer; one-step process of chromatography.
Definition of activity, assay; calculation of yield and specific activity to monitor protein
purification
● Activity – The total units of enzyme in a solution.
o 1 unit of activity is the amount of enzyme causing the transformation of 1 μmol
(10-6) of substrate @ 25°C under optimum conditions.
● Assay – enzyme rate of turning substrates into product… Can usually be observed w/
color changing, radioactive product, antibody binding…
● Yield – activity/total protein
● Specific activity – the number of enzyme units/mg of total protein. Can be calculated by:
[activity]/[total protein]
● The goal of protein purification is to maximize yield and increase specific activity! A
protein is considered pure when further purification steps fail to increase specific activity.
SDS PAGE, log molecular weight vs. relative mobility plot can be used to determine the
molecular wt. of the subunits of a protein. SDS gels can monitor purification of a protein.
Chapter 2:
Noncovalent interactions: hydrogen bonds, electrostatic bonds, Van der Waals interactions
● O–H bond = 470 kJ/mol; O---H bond = 23 kJ/mol
● Noncovalent interactions are relatively weak. They are reversible at body temperature,
making them dynamic. The sum of many of these interactions is strong, as they hold
together proteins, DNA, RNA, and lipids.
● H-bond forms between covalently bonded O-H or N-H (electronegative atoms) and
adjacent H in a similar bond. H-bonds are strengthened by direction (linear H-bond is
stronger than an angled bond).
● Electrostatic bonds/salt bridges/ionic interactions: hydration shells block/mask charges
b/t ions, resulting in a decreased force…
● Van der Waals interactions result from transient dipole-induced dipole interactions of
electron clouds. ~4 kJ/mol. Tokay gecko has thousands of fine hairs that stick to walls via
Van der Waals.
Be able to do calculations with pH, Henderson-Hasselbalch equation; buffers.
● In biochemistry, weak acids and bases are critical. Weak acids and bases partly dissociate.
HA <--> H+ + A-.
● A buffer is a weak acid or base that resists the pH change of a solution. Works best when
[HA] = [A-]. There is usually a window for buffer area of +/- 1 pH.
● Henderson-Hasselbalch: pH = pKa + log{[A-]/[HA]}
● Physiological buffers include phosphate (intracellular buffer) and bicarbonate
(extracellular buffer)
Chapter 3:
Be able to draw the structures of the 20 amino acids at pH 7.0; know the three-letter codes and
one-letter codes for the amino acids. Know which ones are acidic, basic, hydrophilic, and
hydrophobic. L stereoisomers.
Be able to use the Henderson-Hasselbalch equation in calculations on amino acids.
Structure of the peptide bond
● Such a bond is formed by the removal of water
(dehydration) from the α-carboxyl group of one
amino acid and the α-amino group of another.
Peptide nomenclature, N- and C-terminal; disulfide bond
, Purification of proteins:
Column chromatography:
● Ion-exchange: separation by net electric charge of proteins at a given pH.
o Cation-exchange chromatography – solid matrix has negative charge.
o Anion-exchange chromatography – solid matrix has positive charge.
● Size-exclusion – Gel filtration. A molecular sieve of porous polymer beads separates
protein molecules according to their size. The beads have pores and cavities of a
particular size. Larger proteins cannot enter these cavities and take a shorter route path
thru the column.
Affinity chromatography (His-tag and Ni NTA column);
● Based on binding affinity. The beads in the column have a covalently attached chemical
group called a ligand – a group of molecule that binds to a macromolecule such as a
protein. This one-step separation process binds proteins with affinity to the ligand and
retards their movement. After the proteins that do not bind are washed thru the column,
the bound protein is eluted by a solution containing a high conc. of salt or free ligand.
● His-tag (engineered protein w/ a histidine tag)
● Nickel nitrilotriacetate (NiNTA). Nickel 2+ can bind proteins w/ multi-histidine tag. Wash
protein w/ histidine or imidazole containing buffer; one-step process of chromatography.
Definition of activity, assay; calculation of yield and specific activity to monitor protein
purification
● Activity – The total units of enzyme in a solution.
o 1 unit of activity is the amount of enzyme causing the transformation of 1 μmol
(10-6) of substrate @ 25°C under optimum conditions.
● Assay – enzyme rate of turning substrates into product… Can usually be observed w/
color changing, radioactive product, antibody binding…
● Yield – activity/total protein
● Specific activity – the number of enzyme units/mg of total protein. Can be calculated by:
[activity]/[total protein]
● The goal of protein purification is to maximize yield and increase specific activity! A
protein is considered pure when further purification steps fail to increase specific activity.
SDS PAGE, log molecular weight vs. relative mobility plot can be used to determine the
molecular wt. of the subunits of a protein. SDS gels can monitor purification of a protein.
