Lecture 3: pH & Amino Acids Lecture 4: Proteins Primary Structure

Week 2

If the pH is less than the pKa, how will the R group exist?

protonated because there will be a lot of H+ in the acidic environment

If the pH is greater than the pKa, how will the R group exist?

deprotonated because there will few H+ in the basic environment

what does pKa measure

how easily a molecule gives a proton

pH equation

pH = -log[H⁺]

if [H+] = 1M then the pH is ...

0

if [H+] = 10^-7 M then the pH is …

7

pKa equation

pKa= -logKa

henderson hasslebach eqxn

pH = pKa + log([A^-]/[HA})

• works only for weak acids and bases

• for calculating ratio of charged/to uncharged side chains, always put the deprotonated form in the numerator and the protonated form in the denominator.

every weak acid is a ____

buffer

• every buffer is also a weak acid

weak acids are effective buffers at pH values near their …

pKa

• ± 1 unit

How buffering works…

1) start w/HA (weak acid)

2) add OH^- (strong base) to convert HA to A^- bc OH^- takes it’s H⁺

3) when 50% of the OH^- is added, the pH = pKa and [HA] = [A^-]

4) near pKA, curve is relatively flat so pH is relatively unaffected by base (or acid)

HA is a ____ acid

weak

a strong acid converts ___ to ____

A^- to HA

A strong base converts ____ to ___

HA to A^-

Blood Plasma Buffering

CO₂ (g) ⇌ CO₂ (dissolved) ⇌(H₂O added or leaves) H₂CO₃ ⇌ H⁺ + HCO₃^-

• H₂CO₃ is carbonic acid, weak acid

• HCO₃^- bicarbonate, conjugate base

• K₁ = [H⁺][HCO₃^-] / [H⁺]

• K₂ = [H₂CO₃] / [CO₂ (D)][H₂O]

• K₃ = CO₂ (d) / CO₂ (g)

Blood Plasma Buffering Exercise Effect

CO₂ (g) ⇌ CO₂ (dissolved) ⇌(H₂O added or leaves) H₂CO₃ ⇌ H⁺ + HCO₃^-

• vigorous exercise ↑ [H+] which ↑ H₂CO₃ which ↑ CO₂ (d) which ↑ Pressure CO₂ (g) causing it to be exhaled

Blood Plasma Buffering Adding Base Effect

CO₂ (g) ⇌ CO₂ (dissolved) ⇌(H₂O added or leaves) H₂CO₃ ⇌ H⁺ + HCO₃^-

• Base like OH^- reacts with H⁺ causing concentration to go down

• to replenish lost H⁺, eq shifts right so reactant concentrations ↓ and [HCO₃^-] ↑

for low pH (acidic), what does amino acid look like

• NH₃⁺

• COOH

for neutral pH what does amino acid look like

• NH₃⁺

• COO^-

for high pH (basic) what does amino acid look like

• NH₂

• COO^-

Henderson-Hasselbalch for negatively charged amino acids

negative charged version = A^-

ex: pH = pKa + log ([Asp^-]/[Asp⁰]

What’s special about histidine?

1) it likes to remain uncharged at neutral pH

• it has a pKa of 6 so when the pH > pKa, it likes to be deprotonated; however some still remains protonated

2) it can also easily take up H+ or give off H+ so it’s a good buffer

Hydrophobic effect

1) Phenylalanine is surrounded by highly oriented H₂O molecules

• ordered VDW hydration shell

2) when Phe breaks free to go to nonpolar solvent, H₂O becomes free to move around

3) ∆S_transfer > > 0 to make ∆G < 0

Edman Degradation

method for removing and identifying one amino acid at a time from the N-terminus of a polypeptide

Is forming a peptide bond favorable?

no ∆G° > 0

• ribosome couples peptide bond formation to ATP hydrolysis, a highly exergonic rxn

is breaking a peptide bond favorable?

yes, but takes very long time w/o enzyme help

Mass Spectrometry

• ESI = electroscopy ionization MS gets ionized peptides into gas phase w/o destroying them

1) digest protein into shorter pieces/peptides useing protease

2) separate and sequence resulting fragments using tandem mass Spec (MS/MS)

trypsin cleaves after

R/K

chymotrypsin cleaves after

F, W, Y

V’8 cleaves after

D/E

cysteine special property

1) weak acid

2) it can make H-bonds & disulfide bonds

isoelectric point

pH at which net electric charge is 0, designated by pI

• pI is mean of two pKa values

cation-exchange chromatography

net + charged moves slower bc stick to - charged beads

size-exclusion chromatography

large proteins exit faster