TA BIOCHEM Review Flashcards
isoelectric point (refers to the whole protein)
-pH at which the net charge of the amino acid is zero
-ALSO roughly 50% aa’s deprotonated and the other 50% are protonated
when pH below the pl the overall charge of the protein is
positive (think H+ acid)
when pH above the pl the overall charge of the protein is
negative (think OH-)
(A-/HA)= 10^(+) or value>1
DEPROTONATED
(A-/HA)= 10^(-) or value<1
PROTONATED
gel filtration chromatography or size exclusion chromatography
larger molecules elute first whereas smaller molecules elute later
ion exchange chromatography has 2 types
-DEAE (weak anion exchanger)
-CM (weak cation exchanger)
ion exchange chromatography interaction strenght with exchange matrix depends on:
-charge density of protein (modulated by pH)
-strength of ions that compete with protein for binding
2 ways to elute
- modulate pH (start with getting protein with most basic pH a positive charge and wash, repeat to get all of the through)
- increase salt concentrations (both +/- interactions) -> will compete for binding and displace bound proteins with smaller overall charge first ie pl closer to the pH e.g. have 3 protein pH’s of 3,5,7 C would wash away first if using a pH of 9
SDS ONLY reducing
reduces disulfide bridges so same protein appears as smaller in reducing but as whole size in non-reducing
delta H>0
when BREAKING bonds
delta H<0
when FORMING bonds
S>0 and H<0
spontaneous at ALL temps (G<0)
S<0 and H<0
spontaneous at low temps (when TdeltaS is small)
H>0 and S<0
spontaneous at high temps (when TdeltaS is large)
S<0 and H>0
non-spontaneous at all T (G>0)
hemoglobin and hemoglobin have similar binding sites BUT
different quaternanry structures
Fe(II) octahedrally coordinated by four nitrogen groups of poryohrin ring, O2 and His F8
Hb
Deoxy-Hb (T-state) -> RELAXED
-energetically favored when NO oxygen
-lower pH stabilizes with salt brideges
-BPG binds central cavity and increases favorability to stay in T-state
Oxy-Hb (R-state) -> TIGHT
-energetically favored when THERE IS oxygen
Cooperativity in Hb
-O2 + Fe(II)
-Fe(II) moves into plane of heme
-HisF8 interacting with Fe(II) pulled up like a lever and moves helix F
-surronding subunits move relative to one another and change pKa of certain amino acids involved in salt bridges needed to stabilize T-state
-protons released in these amino acid groups and T-state salt bridges break (because pH inc.)
-the cavity in the center shrinks but the C2 symmetry remains in the R-state
cooperativity O2
favors R-state
cooperativity H+
favors T-state (dec. pH) RIGHT shift
cooperativity CO2
favors T-state (dec. pH) RIGHT shift
