Tutorial - Week 3 - pH, acid-base, charge of amino acids & peptides Flashcards
Key concept: Describe pH, acid, and base
- The pH of a soluWon indicates its acidity or basicity
- Acid is a molecule (or group) that donates H +
- Base is a molecule (or group) that accepts H +
Key concept: Describe pKa and isoelectric point (pl)
- pKa is the pH at which the H + dissociates
- Acids have a low pKa
- Bases have a high pKa
- pI, isolectric point: pH at which the net charge is zero
Key concept: Describe acid-base properties of amino acids
- Amino acids have at least 1 acid group (COOH)
& 1 base group (NH 2 ) - The side chain of polar charged amino acids
has one ionisable group
Key concept: Describe how to calculate the charge of individual amino acids
- Charge of amino acid depends on the pH and pKa of
ionisable groups - pKa differ for each amino acids
- pKa (COOH) is < 7.4, negative at pH 7.4
- pKa (NH 3+ ) is >7.4, positive at pH 7.4
- pKa of the side chain of polar charged amino acids
Key concept: Describe how to calculate the charge of a peptide/protein
- Charge of peptide/protein depends on the pH
and pKa of the residue side chains, N-terminus
and C-terminus - N-terminus group (NH 3+ ) is positive at pH 7.4
- C-terminus (COO - ) is negative at pH 7.4
Label each category
T/F: Biomolecules can be very sensitive to pH
True
As the pH of a solution changes, what may also vary?
The charge
What does the charge of molecules, such as in proteins, have a profound effect on?
How they work or if they are going to function
The pH of a solution indicates the…
Acidity or the basicity
What does pH correlate with in a solution?
the H+ (hydrogen ions)
ph = ? (equation)
-log [H+]
[H+] = ? (equation)
10^-pH M
pOH = ? (equation)
-log [-OH]
pOH = ? (equation)
-log [OH-]
pH + pOH = ?
14 - The sum of pH and pOH is always 14. This is because the product of proton concentration and hydroxide concentration must always equal the equilibrium constant for the ionization of water
What does a low pH indicate in terms of how acidic or basic a solution is and which ion is most prominent?
High H+ ions and acidic (pH < 7)
What does a high pH indicate in terms of how acidic or basic a solution is and which ion is most prominent?
Basic (pH > 7) and high OH- ions
What is the neutral pH? What are the values for OH- and H+?
pH = 7 and OH- = H+
What is the value for physiological pH?
7.4 (close to neutral)
Inside cells how low do some compartments get in pH?
4.5-6
Do strong acids dissociate in water? Why?
Yes
1 mol of HCl in water will dissociate and add 1mol of H+ and 1 mol of Cl- to the water
Do strong bases dissociate in water? Why? What gets added to the water?
What will happen to 1 mol of NaCl?
Yes
Dissociates due to unstableness of the molecules due to the strong charges pulling the molecules apart.
1 mol of NaCl will dissociate into 1 mol of OH- and 1 mol of Na+ in the solution
Do weak acids and bases dissociate in water?
Why are they important?
No
Important as they help to keep the concentration of H+ stable and therefore help to keep the pH of the solution relatively constant
How can the ionisation of a weak acid/base in water be expressed?
By an “equilibrium constant, Ka” <—- a is below the K in the notation
Write the equilibrium constant calculation for acetic acid (the acid present in vinegar) as shown in the graphic
What does the pKa express?
The strength of the base or acid
The stronger the acid the ________ its pKa
Smaller
The stronger the base the ________ its pKa
Larger
What is titration?
A titration is a technique where a solution of known concentration is used to determine the concentration of an unknown solution. Typically, the titrant (the know solution) is added from a buret to a known quantity of the analyte (the unknown solution) until the reaction is complete.
How do you calculate the pKa of an acid?
What is the Henderson-Hasselback equation and what does it predict?
The Henderson-Hasselbalch equation can be written as: pH = pKa + log10 ([A–]/[HA]) Where [A–] denotes the molar concentration of the conjugate base (of the acid) and [HA] denotes the molar concentration of the weak acid.
it predicts the response of the
weak acid/base in response to changes to H + or OH -
The ____________________________ of any weak acid is described by the Henderson-Hasselbach equation
shape of the titration curve
What does the following version of the Henderson-Hasselbalch equation allow you to calculate?
Ionisation of a weak acid:
What does the following version of the Henderson-Hasselbalch equation allow you to calculate?
Solve for [H + ]:
What does the following version of the Henderson-Hasselbalch equation allow you to calculate?
Negative log in both sides:
What does the following version of the Henderson-Hasselbalch equation allow you to calculate for?
Substitute pH for –log [H + ] and pKa for –log Ka , and invert –log [HA]/[A-]:
What does the following version of the Henderson-Hasselbalch equation allow you to calculate for?
When [HA] = [A-]:
What is the buffering region in a pH/Percent titrated graph?
What is the pH of the buffering region around?
The pKa of acetic acid is 4.76, what is it’s buffering reqion?
- range of pHs at which addition of OH - , or H + does not change much the overall pH
1.2 (Range of pH at which a molecule (e.g. buffer) can be used to ‘hold’
the pH)
- Buffering region: pH around pKa +/- 1
- pKa of acetic acid is 4.76, its buffering region is pH 3.76-5.76
Define an acid
a molecule (or group) that behaves as a H+ donor
Define a base
a molecule (or group) that behaves as a H+ acceptor
Complete the equation
Complete the equation
Name the group
Name the group
Name the groups
How many standard amino acids are there?
20
What are the defining features of an amino acid?
They have a primary amino group and a carboxyl group bounded to
the same carbon atom (a-Carbon)
At physiological pH they are ____________
What do they contain?
zwitterions- they
contain both positive and negative charges.
Label the features
What do amino acids differ in the structure of?
Their side chains
Amino acids are classified based on the chemistry of the R group
(side chain):
What are the different types of sidechain groups?
- Non-polar or hydrophobic
- Uncharged Polar
- Charged Polar
- Acidic (-)
- Basic (+)
Amino acids dissolved in water at physiological pH (7.4)
exist as ___________ (they carry both ___________
and ____________ (groups)
zwitterions
negatively-charged
positively-charged
Which group in an amino acid is the acceptor and which is the donor? What do they donate and accept? What forms are they in? And what charge is on them?
Amino group is an acceptor of H + , and is in its protonated form (NH 3+ ): PosiFvely-charged
Carboxylic acid group is a H + donor and is in the carboxylate form (COO -): NegaFve-charged
Amino acids are _____________: they donate and accept H+ they serve as an acid or a base
amphoteric
Does an amino group have basic or acidic properties? Is it an acceptor or donor and of what?
Amino group is an acceptor of H + , it has basic properties
Does a carboxylic acid group have acidic or basic properties? Is it a donor or acceptor and of what?
Carboxylic acid group is a H + donor, it has acidic properties
Complete the following
All the amino acids have at least ____ dissociable H+ (NH 3+ & COOH)
two
T/F: Some amino acids also have a dissociable H+ in the side chain
True
Dissociation of ionisable groups is dependent on…?
The pH
At low pH amino acids are what? What form? What charge?
Acidic, lots of H +. NH 3+ and COOH are protonated, charge +1
What happens to an amino acid as the pH increases? What is the first to dissociate?
As the pH increases, COOH is the first to dissociate, Gly becomes globally neutral (zwitterion)
What happens to amino acids as pH increases further?
With further increase at high pH (basic pH, lots of OH -), NH 3+ dissociates and Gly becomes negative -1
What is pI? Describe it
pI = isoelectric point, is the pH at which the net charge is 0 (iso = equal)
pKa = ?
pH at which the H + dissociates: the lower the pKa, the stronger the acidic
properties of the group
pI = formula?
(pK 1 + pK 2 )/2 = 5.97
What about if the amino acid also has an
ionisable side chain (R) (glutamic acid)?
Glutamic acid is negatively-charged at physiological pH
What about if the amino acid also has an
ionisable side chain (R) (histidine)?
pI = (pK R + pK 2 )/2 = 7.59
Overall charge at physiological pH = 0
BUT it is posiUvely-charged at pH below 6
Some cellular organelles have an acidic pH (4.5-6)
The net ________ of an amino acid depends on the ________________
charge
pH inside the cell
The _______ of the amino acid ___________________ is relevant for the overall charge,
________, _________ of the protein
charge
side chain within a protein
structure, function
___ helps us predict the overall _______ of a polypeptide chain/protein at a given pH
pI
charge
The charge of a protein is important to employ biochemistry tools used to characterise,
_________, _________ & ________peptides/proteins
isolate, separate & quantify
Ion-exchange chromatography isolates ions based on their?
Charge
What are two methods that we use in order to separate and isolate proteins using biochemical techniques?
Ion-exchange chromatography and electrophoresis
At physiological pH what state are the carboxylic groups and amino groups in? Why?
Carboxylic groups are negatively charged due to having a pK1 around 2 (below physiological pH of 7) therefore they lose/donate their proton and become negatively charged at pH 7 - are in the IONISED FORM (COO-)
Amino groups have a pKa of around 9-11 and therefore at physiological pH they are positively charged as they have accepted/gained a proton - Are in the PRONATED FORM (NH3+)
When calculating the charge of an AA at physiological pH what do we need to check?
The charge of the sidechains
What is a basic side chain?
Basic side chain (e.g. Lys has a protonated amino group NH 3 +):
pK R (R below the pK), is the pH at which the side chain becomes neutral
What is an acidic side chain?
Acidic side chain (e.g. COOH, which tends to lose H+):
pK R, is the pH at which the side chain becomes negatively-charged
What are the green and orange boxes highlighting?
Green = basic side chain pKr values and orange = acidic side chain pKr values
Calculate and explain
Lysine: At pH 1, COOH doesn’t lose/donate it’s proton until pH above it’s pKa of 2.18 therefore stays neutral. However, the amino group and R group have pKas or above 1 therefore they gain protons as they become pronated due to being below their pKa or 8.95 and 10.53.
The above process is the same for all the different pHs. If the pH is above the pKa of the carboxylic group then the group will become ionised and lose a proton. If the pH is below the pKa of the amino group then the amino group will become protonated and gain a proton.
The carboxylic group cannot ever gain, and the amino group can never lose a proton.
The R groups can gain or donate depending on if the pH is below or above it’s pKa, respectively
If the pH is above the pKa of the carboxylic group of an AA what happens to the carboxylic group?
It loses/donates a proton and therefore becomes ionised with a charge (for that group) of -1
If the pH is below the pKa of the amino group of an AA what happens to the amino group?
It gains/accepts a proton and therefore becomes protonated with a charge (for that group) of +1
For histidine - Does an R-group become pronated or ionised?
It can become protonated/gain an electron. If the pH is below it’s pKa then it gains/accepts a proton. If the pH is above it’s pKa then it remains neutral and does not donate a proton
How do you calculate the charge of an AA at any given pH?
You look at the pKas for the different groups, and calculate the charge of each group, add the charges, and then that is your overall charge for the amino acid
How do you determine if a sidechain can be negatively or positively charged?
You look at the components of the side chain.
If it contains a carboxylic group on the end then it can be either neutral or negatively charged at -1 (ionised).
If it contains an amino group on the end of the R-group then it can be either neutral or positively charged at +1 (protonated)
How do proteins form peptide bonds?
They use the carboxylic acid group and the amino group between two proteins to form the peptide bonds
Where do the only charges that exist within a protein or peptide come from?
Come from the termini (N-terminus and C-terminus) - the carboxylic acid group in one end and the amino group in the other end
The other thing that determines charge is the sidechain which is ionisable
At pH 7 what are the charges on the two termini?
N-terminus = Positive charge (amino group - NH3+)
C-terminus = Negative charge (carboxylic acid group - COO-)
What is the charge of this protein?
charge = 0
Because at physiological pH of 7, N-terminus (amino group) is +1, C-terminus (carboxylic acid group) is -1. Then you look at the sidechain and since this sidechain doesn’t have a charge at all (falls into the category in the table without a charge) then it doesn’t contribute and the overall charge is 0
See image and calculate. How do you calculate? Where do you start?
You look into the amino acids 3-letter-codes and see if any of the proteins have side chains that can become charged
You only look at the termini (both cancel each other out at physiological pH therefore they equate to 0), and then you look at the side chains of all the proteins within the peptide.
You then add all the side chain charges together and calculate from that the overall charge of the peptide - using the table in the image included
You find that the charged amino acids are Glu (-1), Lys (+1) and Arg (+1) residues have charged side chains at pH 7.4
Overall Charge of this peptide at pH 7.4 is +1
Calculate the overall charge of this peptide (use the table of AA charges and their side chains - consider the pH difference and how it effects charge)
