CHE 212

Introduction to Biochemistry
D. BaNdA
email: dannybanda@mu.ac.zm

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 Syllabus
- Course Overview
1. Biophysical chemistry
Properties of water as solvent for biochemical reactions, hydrogen
bonding. Weak acids and bases. PH and Pka determinations, buffers and
their role in biological systems. Physiologically important buffer systems.

2. Biomolecules
The role of carbon, hydrogen, nitrogen, sulfur and phosphorus in
biomolecules

• Proteins
Overview of proteins, structure and properties of amino acids, titration
curves, amino acids as electrolytes. Peptides, structure of proteins,
physical and chemical properties of proteins
Protein separation and purification techniques. Qualitative and
quantities analysis of proteins ……etc. see course outline 2
 - Notes on Grading
Assessment
Continuous Assessment 40%

Assignments 5%
Tests 20%
Laboratory experiments 15%

Final Theory Examinations 60%

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 Biochemistry
This is the chemistry of living things
Biochemistry
– provides fundamental understanding of the
molecular basis for function /malfunction of living
things.
– Provides a broad understanding of the molecular
basis of life.
– Explains what goes wrong to produce disease.

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How does Biochemistry impact you
Medicine

Agriculture

Industrial applications

Environmental applications

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Biophysical Chemistry

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 Lesson objectives
 Describe properties of water as a solvent for
biochemical reactions, hydrogen bonding
 Describe ionization of a water molecule
 Define pH and through calculations, determine pH
values of given solutions using the Henderson-
Hasselbalch equation.
 Define buffers and give examples of physiological
buffers.
 Explain the importance of buffers in living organisms
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 Water
All living things are dependent on water.

Inside the human body, cells are 70 to 95

percent water.
The abundance of water is a major reason
Earth can support life.
Life is dependent on the properties of water

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 The Structure of Water
 two hydrogen atoms
are each joined to an
oxygen atom by a
single covalent bond.

 The key to water's

unusual properties is
that the electrons of
each covalent bond are
not shared equally
between oxygen and
hydrogen atoms.
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 Structure …….
 The unequal sharing of electrons causes the
oxygen end of the molecule to have a slight
negative charge, while the end with the two
hydrogen atoms is slightly positive.
 A molecule in which opposite ends have
opposite electric charges is called a polar
molecule.
 Water is a compound consisting of polar
molecules.

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 Hydrogen bonds
 An unshielded hydrogen nucleus covalently bound to an
electron-withdrawing oxygen or nitrogen atom can interact
with an unshared electron pair on another oxygen or nitrogen
atom to form a hydrogen bond.

 Water molecules are attracted to one another in a specific

way.

 The slightly negative oxygen end of one molecule attracts the

slightly positive hydrogen ends of adjacent water molecules

 The attraction in H-bonds is not as strong as in an ionic bond

because electrons are not transferred
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 H-bonds continued….
 Hydrogen bonding profoundly influences the physical
properties of water and accounts for its exceptionally high
viscosity, surface tension, and boiling point.

 On average, each molecule in liquid water associates through

hydrogen bonds with 3.5 others.

 Hydrogen bonding enables water to dissolve many organic

biomolecules that contain functional groups which can
participate in hydrogen bonding.

 The oxygen atoms of aldehydes, ketones, and amides provide

pairs of electrons that can serve as hydrogen acceptors.
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 Water's Life-Supporting Properties
The polar nature of water and the effects of
hydrogen bonding explain most of water's unique
properties.

These properties include cohesion and adhesion,

temperature moderation, the lower density of ice
compared to liquid water, and water's ability to
dissolve other substances.

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 Cohesion and Adhesion
The tendency of molecules of the same kind to stick
to one another is called cohesion.
Cohesion is much stronger for water than for most
other liquids.
Water molecules are also attracted to certain other
molecules.
The type of attraction that occurs between unlike
molecules is called adhesion.

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 Water's Ability to Dissolve Other
Substances
When water is the solvent, the result is called an
aqueous solution (from the Latin word aqua,
"water").
Water is the main solvent inside all cells, in blood,
and in plant sap.

Water dissolves an enormous variety of solutes

necessary for life due to its polarity and hydrogen
bonding ability.
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 Water is an ideal biologic solvent
 A water molecule is an irregular, slightly skewed tetrahedron
with oxygen at its center

 The 105-degree angle between the hydrogens differs slightly

from the ideal tetrahedral angle, 109.5 degrees.

 Water is a dipole, a molecule with electrical charge

distributed asymmetrically about its structure.

 Water, a strong dipole, has a high dielectric constant

 Its strong dipole and high dielectric constant enable water to

dissolve large quantities of charged compounds such as salts.
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 Interaction of water
Interaction with water influences the structure of
biomolecules

Covalent and non-covalent bonds stabilize

biomolecules.

Though covalent bonds are the strongest force

holding molecules together, non-covalent bonds
make significant contributions to the structure,
stability, and functional competence of
macromolecules in living cells.
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 Ionization of water
The ability of water to ionize, though, slightly is of
central importance for life.
Water is capable of acting both as an acid and a base,
hence its ionization can be represented as below

H2O + H2O → H3O+ + OH-

The ionization produces a hydronium ion and a
hydroxide ion
Like all reversible reactions, the ionization of water
can be described by an equilibrium constant

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 Ionization of water
• The degree of ionization of water at equilibrium is
small; at 25 °C only about two of every 109
molecules in pure water are ionized at any instant.
• The equilibrium constant for the reversible ionization
of water is

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 Ionization of water
In pure water at 25 C, the concentration of water is 55.5 M
divided by its molecular mass (1,000 g/L)/(18.015 g/mol),
We can substitute 55.5 M in the equilibrium constant
expression to yield

Which on rearranging becomes

(55.5M)(Keq) = [H+][OH-]=Kw
Where Kw designates the product of (55.5M)(Keq) , called the
ion product of water at 25oC
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 Continuation….
The value for Keq, determined by electrical-conductivity
measurements of pure water, 1.8 x 10-16M at 25oC.
Substituting the values of Keq, gives the ion product of
water to be
Kw = [H+][OH-] = (55.5M)(1.8 x 10-16M)
= 1.0 x 10-14M2
When there are exactly equal concentrations of H+ and OH-,
as in pure water, the solution is said to be at neutral pH
At this pH, the concentration of H and OH can be calculated
from the ion product of water as follows:

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 pH
pH is the negative log of [H+]. A low pH characterizes an
acidic solution, and a high pH denotes a basic solution.

pH = -log [H+]

The total hydrogen ion concentration from all sources is

experimentally measurable and is expressed as the pH of
the solution.

The ion product of water, Kw, is the basis for the pH

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 Significance of pH
Measurement of pH is one of the most important and
frequently used procedures in biochemistry.
The pH affects the structure and activity of biological
macromolecules; for example, the catalytic activity of
enzymes is strongly dependent on pH.
 Measurements of the pH of blood and urine are commonly
used in medical diagnoses. The pH of the blood plasma of
people with severe, uncontrolled diabetes, for example, is
often below the normal value of 7.4; this condition is called
acidosis.
In certain other disease states the pH of the blood is higher
than normal, the condition of alkalosis. 25
 But……..
• The chemical reactions of life constantly
produce acids and bases within cells. These
have a tendency to throw off the pH values.
We need some sort of mechanism to minimize
how much the pH is altered

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 Questions related to pH
1. What is the concentration of OH– in a solution with a H+
concentration of 1.3×10-4 M?

2. What is the pH of a solution consisting of aqueous ammonia

whose concentration is 1.8 x 10-5.

3. What are the pH values of (a) 2.0 x 10−2 mol/L KOH and of
(b) 2.0 x 10−6 mol/L KOH?

The OH− arises from two sources, KOH and water. Since pH is
determined by the total [H+] (and pOH by the total [OH−]), both
sources must be considered. In the first case (a), the
contribution of water to the total [OH−] is negligible. The same
cannot be said for the second case (b): 27
Question 3 contin…..

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 Weak acids and Bases
Weak acids are acids that partially ionize in water.

The strength of weak acids is expressed by pKa, the

negative log of the acid dissociation constant. Strong
acids have low pKa values and weak acids have high
pKa values

Weak acids are common in biological systems and play

important roles in metabolism and its regulation.

A proton donor and its corresponding proton acceptor

make up a conjugate acid-base pair
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 Weak acids and Bases
 Many biochemicals possess functional groups
that are weak acids or bases.

 Carboxyl groups, amino groups, and the second

phosphate dissociation of phosphate esters are
present in proteins and nucleic acids, most
coenzymes, and most intermediary metabolites.

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 Knowledge of the dissociation of weak acids and bases thus
is basic to understanding the influence of intracellular pH on
structure and biologic activity.

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The Henderson-Hasselbalch Equation
It describes the behavior of weak acids and
buffers.
It is derived from the ionization of a weak
acid.
HA ↔ H+ + A-

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 Henderson- Haselbalch Equation
 It has great predictive value, e.g at half
neutralization i.e. [HA] = [A-],
pH= pKa
 pH tends to be greater than pKa i.e. move
towards alkalinity when [A-] is higher than the
acid i.e. pKa + x value
 pH tends to be lower than pKa i.e. move
towards acidity i.e. pKa – x value.

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 Review question
Q1. Calculate the pH of a buffer solution
containing 0.500mol dm-3 ethanoic acid and
0.100 mol dm-3 sodium ethanoate.
Ka = for ethanoic acid = 1.74 x 10-5 moldm-3

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Q2. What is pH of the solution prepared by mixing 50 mL of
0.01 M acetic acid and 50 mL of 0.01 M sodium acetate?

Solution
The concentrations of the acid and conjugate base are identical, thus
their ratio is

and after plugging it into the Henderson-Hasselbalch equation

we have pH=pKa (which is 4.75 for the acetic acid).

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Let's try another example:

Q3. How much NaOH must be added to 50 mL of 0.1 M acetic acid to

prepare pH 5.0 buffer?
Plugging given pH and known pKa into the Henderson-Hasselbalch
equation we get

and after simple rearranging

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• Now it is just a matter of stoichiometry - added NaOH neutralizes
acid and produces acetate ion.
• Initially there was 0.05 L×0.1 M=0.005 moles of acetic acid, after
adding n moles of NaOH there will be 0.005-n moles of acetic
acid and n moles of acetate (this is pretty easy, as they react 1:1,
we also assume NaOH is a limiting reagent).
• If so, concentrations of HAcetate and Acetate- are

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 Plug the two formulae into the Henderson equation…

This equation can be easily solved for n, which is 0.00320

moles - and that's the final answer to the question.

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• This is almost identical problem, with one important difference -
we deal with a triprotic acid.

pKa values for phosphoric acid are 2.15, 7.20 and 12.35 respectively,
of those the second one is very close to the pH we need.

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 Practice Questions

1. What volume of 1 M HCl must be added to 100 mL of 0.1 M

ammonia to prepare pH 10.0 buffer?

2. How much HCl has to be added to 50 mL of 0.1 M sodium

acetate to prepare pH 5.0 buffer?

3. How to prepare 200 mL of pH=4.5 0.1 M acetate buffer from 2 M

acetic acid and 1 M sodium acetate?

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 Physiological Buffers
• Solution containing equal concentrations of an acid and its
conjugate base.

• Buffers have the ability to resist a change in pH following

addition of strong acid or base.

• Many metabolic reactions are accompanied by the release or

uptake of protons, hence most intracellular reactions are
buffered.

• Maintenance of a constant pH involves buffering by

phosphate, bicarbonate, and proteins, which accept or
release protons to resist a change in pH
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 Physiologic buffers continu…….
• Buffers resist a change in pH when protons are
produced or consumed.

• Maximum buffering capacity occurs ± 1 pH unit on

either side of pKa.

• Physiologic buffers include bicarbonate,

orthophosphate, and proteins.
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 Laboratory Buffers
• For experiments using tissue extracts or enzymes,
constant pH is maintained by the addition of buffers
such as
i. MES ([2-N-morpholino]ethanesulfonic acid, pKa 6.1),
ii. inorganic orthophosphate (pKa2 7.2),
iii. HEPES (N-hydroxyethylpiperazine-N9-2-
ethanesulfonic acid, pKa 6.8), or
iv. Tris (tris[hydroxymethyl] aminomethane, pKa 8.3).

• The value of pKa relative to the desired pH is the major

determinant of which buffer is selected. 46
 Importance of Buffers in Physiological
Systems:
 Processes that take place in living organisms are called physiological
processes. Like blood circulatory system, respiration etc.

 The internal pH of most living cells is close to 7.0. The pH of human

blood is 7.4. A blood pH of below 7 or above 7.8 can cause death
within minutes.

 So buffering of blood pH is very important to stabilize it around 7.4.

 pH plays an important role in almost all biological processes. Small

change in pH or high pH can cause metabolic implications in human
body like acidosis and alkalosis.
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Bicarbonates buffers (Buffering in
blood)

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 Bicarbonate buffer…..
• Carbonic acid concentration is controlled by respiration through
lungs while hydrogen carbonate concentration is controlled by
urination through kidneys.

• Carbonic acid buffer system is a critical buffer for blood as in the

absence of this buffer system the pH may fall below this normal
value within blood producing a condition called acidosis ( acidosis
may be respiratory or metabolic acidosis)

• or the pH may rise above normal level producing a condition known

as alkalosis (alkalosis may be respiratory or metabolic acidosis).

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Phosphate buffer

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 Protein buffer (Buffering in Cells and
Tissues)
• Proteins are mainly composed of amino acids.
•
• These amino acids contain functional groups that act as
weak acid and bases when there are sharp changes in pH in
order to stabilize the pH within the body cells.

• Protein is a significant buffer

• the main buffering site for protein is cells and tissues but
even in blood it acts as a buffer consuming hydrogen ions
producing due to the dissociation of the carbonic acid into
hydrogen bicarbonate. 52
• To understand the proteins as a buffer we have to
look into the structure of amino acids which consists
of

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 THE END
QUESTIONS?

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