BCH2333 – Introduction to Biochemistry

Lecture 3

Acids, Bases, pH

Buffers

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Covalent ~ 150 to 400 kJ/mol
Types of Non-covalent Interactions In Biochemistry:
Approx. Energy
(kJ/mol)

Charge-Charge
20-80

O O

van der Waals

2-4

H-Bond
8-20

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Acid-Base Equilibria In Biochemistry:

Extracellular

Aqueous
(pHA) Biochemical environment consists of
X X+ aqueous regions (extracellular,
intracellular compartments) separated
by hydrophobic regions (membranes).

Hydrophobic Molecular interactions and behaviour in

biochemistry rely on the ionization state
of a biomolecule (or building block!)

Intracellular The pH of the environment is tightly

Aqueous regulated, and will determine the
(pHB) equilibrium position of ionization states
X X+ of a given species

This is all governed by water!

Ionization is controlled to permit particular functions
(e.g. trans-membrane transport)
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Acids & Bases – Review:

In biochemistry, acids can be thought of by Brønsted-Lowry definition:

Acid = proton donor Base = proton
acceptor
Acids and bases can be further classified by their strength:

Strong: Weak:
Strong acids/bases nearly Weak acids/bases dissociate partially, resulting in a
completely dissociate in solution measurable equilibrium between the acid-conjugate
base, or base-conjugate acid.

H-A A - + H+ e.g. HCl H-A A - + H+ e.g. Acetic Acid

Conjugate base
Weak conjugate base

B- + H + BH e.g. NaOH B- + H + BH e.g. Carbonate

Weak conjugate acid Conjugate acid
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Ionization Equilibrium of Water:

Water is essentially neutral, but it has a tendency to gain and lose protons (i.e.
ionize)

H2O + H2O H3O+ + -OH

H2O H+ + -OH A proton never exists as H+, it is always

aqueous. When we write H+ in
biochemistry, it really means H3O+
Recall equilibrium constants:
Activity (a) = an effective concentration
that depends on its surroundings.
For the dilute concentrations reached
in biochemistry, ax≈[X].

Neutral

So, neutral pH = 7 (at 37°C, the pH of water is ≈6.8)

Physiological pH range is ~6.5 to ~8.0
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pH:
pH [H+] a [OH-]
0 (100) 1.0 0.00000000000001 (10-14)
1 (10-1) 0.1 0.0000000000001 (10-13)
2 (10-2) 0.01 0.000000000001 (10-12)
3 (10-3) 0.001 0.00000000001 (10-11)
10x D[H+]
4 (10 )
-4
0.0001 0.0000000001 (10-10)
5 (10-5) 0.00001 0.000000001 (10-9)
6 (10-6) 0.000001 0.00000001 (10-8)
7 (10-7) 0.0000001 0.0000001 (10-7)
8 (10-8) 0.00000001 0.000001 (10-6)
9 (10-9) 0.000000001 0.00001 (10-5)
10 (10-10) 0.0000000001 0.0001 (10-4)
11 (10-11) 0.00000000001 0.001 (10-3)
12 (10-12) 0.000000000001 0.01 (10-2)
13 (10-13) 0.0000000000001 0.1 (10-1)
14 (10-14) 0.00000000000001 1.0 (100 )
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Biochemical Acids and Bases:

Weak acids and weak bases are key for maintaining environmental pH in the face
of changing protonation states of biomolecules

HA H + + -A Monoprotic

Diprotic

A larger Ka (and smaller pKa) is Polyprotic

indicative of a stronger acid
(more propensity to dissociate)

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Quantifying the effect of DpH (Henderson-Hasselbalch):

Derivation:

HA H + + -A Quantitative relationship between the

pH of a solution and the ratio of the
concentration of the deprotonated to
protonated form of an ionizable group.

pH pKa
What is the pH of a mixture of acid and
conjugated base?

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Quantifying the effect of DpH (Henderson-Hasselbalch):

Example:

If the pH of lactic acid solution is 5.2, and the lactate concentration is 8 mM, what is
the concentration of lactic acid? (pKa=3.86)

Antilog: 10x

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Buffers:
When an acid and base are added to solution, there is an interaction of their
dissociated species that shifts their equilibrium positions in line with LeChatelier’s
principle. Monoprotic Acid

Titration HA H + + -A
B
BH+

The shape of the titration curves are

pKa=9.25 similar, they are just translated
vertically depending on the pKa of the
titrated acid.
At the midpoint, [HA]=[A-]

pKa=3.75

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Buffers:
Small DpH occurs with incremental additions of base around the pKa = buffering
As the pH approaches the pKa, a sufficient amount of conjugate acid and conjugate
base are present to complex with newly formed H+ or -OH
Titration HA H + + -A
B
BH+
Neutralization by conjugate base:
HCl Cl- + H+
pKa=9.25
HCOOH HCOO- + H+

Neutralization by dissociated proton:

NaOH -
OH + Na+
pKa=3.75 HCOOH HCOO- + H+

Match desired pH to the pKa of the buffer to

maintain [H+]
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Buffers Calculation Example:

Formic acid is provided (pKa=3.75) to make a 0.1 M buffer at pH=4.00. How
should this buffer be made?
We are given the total concentration
of the buffer is 0.1 M
Eqn 2

Substitute Eqn 1 into Eqn 2:

Eqn 1

To make the buffer, mix 36 mL of 1M formic acid (HA) with 64

mL of 1M sodium formate (A-) and dilute to 1L.
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Buffers & Titration Calculations:

Adding a base (-OH) to a solution will consume the acid (HA)

As –OH is added, HAA-, such that

Titration

x = mole equivalents of base added

vol = total volume of the solution

pKa=9.25
If co = initial [HA], then

pKa=3.75

Equivalents
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Buffers & Titration Calculations:

How many equivalents of NaOH are required to bring a 0.2 M acetic acid buffer
(pKa=4.76) to a pH of 5.4?

0.16 equivalent of NaOH are required.

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Polyprotic Acids:
So far we have dealt with monoprotic acids: can lose only one proton

Many biological acids/bases have more than one proton to lose: polyprotic acids
e.g. Oxalic Acid Succinic Acid
pKas of polyprotic
acid/base groups are
not independent
Better
delocalization
of initial
pKa1=1.2 pKa1=4.2 The effect of one
charge acid/base group on
another depends on the
DpKa=3 DpKa=1.4 distance between the
Better groups (i.e. charge
separation of separation)
anionic
pKa2=4.2 pKa2=5.6
centres after
2nd H+ loss The close approach of
like charges in second
deprotonation step is
destabilizing!
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Titration of Polyprotic Acids:

Recall: Midpoint = pKa

Equivalence point: point at

which chemically equivalent
quantities of acid and base
pKa3
have been mixed

Blue region indicates buffering

region ideal for biochemistry;
pKa2 enabled by monobasic-dibasic
equilibrium

pKa1

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Buffers example – Bicarbonate in the blood:

Blood pH is a readout of the pH of tissues, such that deviations from normal range
(7.35 to 7.45) can indicate pathology.
pH<7.35 = acidosis pH>7.45 = alkalosis
Keq1 Keq2
Metabolic product
constantly produced CO2 + H2O H2CO3 H+ + HCO3-
and expelled by
Anhydration Dissociation
breathing

Solve for HCO and substitute:

- The CO2/HCO3- system is
3
the key regulatory system
for blood pH (homeostasis)
Gather constants:
pKa=6.1
55.5 M
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Buffers example – Bicarbonate in the blood:

The equilibrium position of bicarbonate is also important for CO2 transport from site of
production to site of excretion (lungs).

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Blood buffers and buffering capacity:

In addition to the HCO3- system, blood contains phosphate buffer and serum proteins
(e.g. albumin).
Which on is the key player? Comes down to buffering capacity, determined
by the individual concentrations of A- and HA

Buffering capacity is a measure of how

much titrant (or acid) can be consumed by
the buffer system. The more buffer
component mass that exists, the more
titrant it can consume.

[HCO3-] = 22 to 29 mM

[H2PO4] = 0.8 to 1.5 mM

[Albumin] = 0.54 to 0.74 mM

Proteins contain ionizable
groups that act as acids and What is it about proteins that allow them to act
bases (more to come…) as buffers?
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Proteins as buffers:
Some biomolecules are polyprotic, with one proton site being acidic and one proton
site being basic
These molecules are known as ampholytes, and they have both an
acidic and a basic pKa
Glycine Net Charge
Less Acidic More Acidic

-1

Predominant form
Isoelectric point (pI): of glycine at
0 When biomolecule net physiological pH
charge is zero
Zwitterion (net q=0)

At the pI, the predominant form is the zwitterion.

However non-zwitterionic states exist but they are at
+1 equal concentrations, maintaining a net charge of 0.
@pI, [-1]=[+1]
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Proteins as buffers:
The distribution of species in solution is directly dependent upon environmental pH

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Quantitation of pI:
The pI can be quantified by the Henderson-Hasselbalch equation:
-1 +1
The average of the pKas relating to the
ionization of the zwitterion

Glycine Net Charge

Aspartic Acid Net Charge

-1

-2
0

-1
0
+1
+1

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pI of Proteins (polyampholytes)
Proteins contain up to hundreds of pH-responsive groups; when the net charge of the
protein is 0, anions balance cations and the pI is reached

Three conclusions to be applied to proteins and pI:

@ pH<pI, |S(q-)|<|S(q+)|, @ pH=pI, |S(q-)|=|S(q+)|, @ pH>pI, |S(q-)|>|S(q+)|,

net charge=+ve net charge=0 net charge=-ve

=anionic =cationic
This has a major impact on protein solubility and
biomolecular interactions: why?
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pI of Proteins (polyampholytes)
This has a major impact on protein solubility and biomolecular interactions: why?

Non-covalent intermolecular interactions

dictate the function of even large
biomolecules.
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pH Extra Resources:

https://www.khanacademy.org/science/chemistry/oxidation-reduction/acids-and-bases/v/pka-and-pkb-
relationship

http://yeahchemistry.com/tutorials/Acid-Base

http://mysite.science.uottawa.ca/aflynn/Organic_Acid-Base.html

https://www.mcb.ucdavis.edu/courses/bis102/acid-base/

Buffers Extra Resources:

http://chemcollective.org/activities/tutorials/buffers/buffers1

https://www.khanacademy.org/science/chemistry/oxidation-reduction/acids-and-bases/v/buffers-and-
hendersen-hasselbalch

https://www.youtube.com/watch?v=MlH-qE-Xki8

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Amino Acid Structures & pKas:

2 (1.8 – 2.5)
4.0 (3.9-4.1)
6
8.4
10.5
9.5 (9.1 – 10.7)
10.5
12.5

Amino acid memory game

courtesy of Dr. Musgaard on
Brightspace

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DNA Base Structures:

(in RNA)

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