LANDMARK UNIVERSITY, OMU-ARAN

BCH 212LECTURE NOTE

INTRODUCTION TO PHYSICAL BIOCHEMISTRY

DEC, ‘12
 LECTURE NOTE FOR BCH 212

Water is essential for life. Life as we know exist because of water, a common component of all
biological cells and their extracellular environment. It covers 2/3 of the earth's surface and every
living thing is dependent upon it. Water is the most abundant constituent of the human body
accounting approximately 60 to 70% of the body mass in a normal adult and it is a major
component of many bodily fluids including blood, urine, and saliva. An understanding of water
and its properties is important to the study of biochemistry. The macromolecular components of
cells—proteins, polysaccharides, nucleic acids, and membranes—assume their characteristics
shapes in response to water. Some types of molecules interact extensively with water and as a
result are very soluble. Other molecules do not dissolve easily in water and to associate with
each other to avoid water. Much of the metabolic machinery of cells has to operate in an aqueous
environment because water is an essential solvent as well as a substrate for many cellular
reactions.

The physical properties of water allow it to act as a solvent for ionic and other polar substances
and the chemical properties of water allow it to form weak bonds with other compounds,
including other water molecules. The chemical properties of water are also related to the
functions of macromolecules, entire cells and organisms. The Interactions are important sources
of structural stability in macromolecule and large cellular reactions. It is important to in mind
that mind that water is not just an inert solvent; it is also a substrate for many cellular reaction.

IMPORTANCE OF WATER

1. It is a medium in which body solutes, both organic and inorganic, are dissolved and metabolic
reaction take place.

2. It acts as a vehicle for transport of solute.

3. Water itself participates as a substrate and a product in many chemical reactions, e.g.
glycolysis, citric acid cycle and respiratory chain.

4 .The stability of subcellular structures and activities of the numerous enzymes are dependent
on adequate cell hydration.

5. The abundance of water in cells and tissues of all large multicellular organisms regulate the
temperature because of water‘s highest latent heat of evaporation.

6. Water acts as a lubricant in the body so as to prevent friction in joints, peritoneum and
conjunctiva.

7. Both a relative deficiency and an excess of water impair the function of tissues and organs
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 THE WATER MOLECULE IS POLAR

A water molecule (H2O) is V-shaped and the angle between the two covalent (O-H)
bonds is 104.5o. Some important properties of water arise from its angled shape and the
intermolecular bonds that it can form. An oxygen atom has eight electrons in the inner
shell and six electrons in the outer shell. The outer shell can potentially accommodate
four pairs of electrons in one s orbital and three p orbitals. However, the structure of
water and its properties can be better explained by assuming that the electrons in the
outer shell occupy four sp3 hybrid orbitals. Think of these four orbitals as occupying the
four corners of tetrahedron that surrounds the central atom of oxygen. Two of the sp3
hybrid orbitals contain a pair of electrons and the other two each contain a single
electron. This means that oxygen can form covalent bonds with other atoms by sharing
electrons to fill these single electron orbitals. In water the covalent bonds involve two
different hydrogen atoms each of which shares its single electron with the oxygen atoms.

The H-O-H bond angle in free water molecule is 104.5o but if the electron orbitals were
really pointing to the four corners of a tetrahedron, the angle would 109.5o The usual
explanation for this difference is that there is a strong repulsion between the lone electron
pairs and this repulsion pushes the covalent bond orbitals closer together, reducing the
angle from 109.5o to 104.5o.

Oxygen atoms are more electronegative than hydrogen atoms because an oxygen nucleus
attracts electron attracts electrons more strongly than the single proton in the hydrogen
nucleus. As a result, an uneven distribution of charge occurs within each O-H bond of the
water molecule with oxygen bearing a partial negative charge and hydrogen bearing a
partial positive charge. This uneven distribution of charge within a bond is known as a
dipole and the bond is said to be polar.

The polarity of a molecule depends on the polarity of its covalent bonds and it geometry.
The angled arrangement of the polar O-H bonds of water creates a permanent dipole for
the molecule. Water and gaseous ammonia are electrically neutral, both molecule are
polar. The high solubility of polar ammonia molecules in water is facilitated by strong
interactions with polar water molecules. The solubility of ammonia in water demonstrates
the principle that ―likes dissolves likes‖.

HYDROGEN BONDING IN WATER

One of the most important consequences of the polarity of the water molecule is that water
molecules attract one another. The attraction between one of the slightest positive hydrogen
atoms of one water molecule and the slightest negative electron pairs in one of the sp3 hybrid
orbitals produces a hydrogen bond. In a hydrogen bond between two water molecules, the
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hydrogen atom remains covalently bonded to its oxygen atom, the hydrogen donor. At the same
time, it is attracted to another oxygen atom called the hydrogen acceptor. In effect, the hydrogen
atom is being shared (unequally) between the two oxygen atoms. The distance from the
hydrogen atom to the acceptor oxygen is about twice the length of the covalent bond.

Water is the only molecule capable of forming hydrogen bonds; these interactions can occur
between any electronegative atom and hydrogen attached to another electronegative atom.
Hydrogen bonds are much weaker than typical covalent bonds. The strength of hydrogen bonds
in water and in solution is estimated to be about 20 kJ mol‾¹

H—O—H + H—O—H → O—H-----O ∆Hƒ -20 kJ mol‾¹

Fig. 1 Hydrogen Bonding

About 20 kJ mol‾¹ of heat is given off when hydrogen-bonded water molecules form in water
under standard conditions (standard conditions are 1atm pressure and a temp 25°C). This value is
the standard enthalpy of formation (∆Hƒ). It means that the change in enthalpy when hydrogen
bonds form is about - 20 kJ mol‾¹ per molecule of water. This is equivalent to saying that +20 kJ
mol‾¹ of heat energy is required to disrupt hydrogen bonds between water molecules—the
reverse of the reaction above. The strength of hydrogen bond is less than 5% of the strength of
typical covalent bonds. Hydrogen bonds are weak interactions compared to covalent bonds but
their large number is the reason for the stability of liquid water.

Orientation is important in hydrogen bonding. A hydrogen bond is most stable when the
hydrogen atom and the two electronegative atoms associated with it (the two oxygen atoms, in
the case of water) are aligned, or nearly in line

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 Figure: Hydrogen Bonding in Water

Water molecules are unusual because they can form four O—H—O aligned hydrogen bonds
with up to four other water molecules. They can donate each of their two hydrogen atoms to two
other water molecules and accept two hydrogen atoms from two other water molecules. Each
hydrogen atom can participate in only one hydrogen bond. This tetrahedral lattice structure is
responsible for the crystalline structure of ice. In the common form of ice, every molecule of
water participates in four hydrogen bonds, as expected. Each of the hydrogen bonds points to the
oxygen atom of an adjacent water molecule and these four adjacent hydrogen bonded oxygen
atoms occupy the vertices of a tetrahedron. This arrangement is consistent with the structure of
water shown in Figure 2, except that the bond angles are all equal (109.5°). This is because the
polarity of individual water molecules, which distorts the bond angles, is cancelled by the
presence of hydrogen bonds. The ability of water molecules in ice to form four hydrogen bonds
and the strength of these hydrogen bonds give ice unusually high melting point because a large
amount of energy, in form of heat is required to disrupt the hydrogen bonded lattice of ice. In the
transition from ice to water, only some hydrogen bonds are broken. Liquid water has a rapidly
changing structure as hydrogen bonds break and new bonds form; the half-life of hydrogen
bonds in water is less than 1x 10‾¹¹ s. Thus the structure of liquid water is constantly fluctuating
with a variety of structures containing many water molecules being constantly formed and
changed, this responsible for the fluidity of water. The fluidity of liquid water is primarily a
consequence of the constantly fluctuating pattern of hydrogen bonding as hydrogen bonds break
and reform.

The density of most substances increases upon freezing as molecular motion slows and tightly
packed crystals form. Water expands as the temperature drops below 4oC. This expansion is
caused by the formation of the more open hydrogen –bonded ice crystal in which the water
molecule.

Two additional properties of water are related to its hydrogen-bonding characteristics—its

specific heat and it heat of vaporization. Relatively large amount of heat is required to raise the
temperature of water because each water molecule participates in multiple hydrogen bonds that
must be broken in order for the kinetic energy of the water molecule to increase. The abundance
of water in the cells and tissues of all large multicellular organisms means that temperature
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fluctuations within the cells are minimized. This feature is of critical biological importance since
the rates of most biochemical reactions are sensitive to temperature.

HYDROGEN BONDS AND OTHER WEAK INTERACTIONS INFLUENCE

BIOLOGICAL MOLECULE

Biochemists are concerned not just with strong covalent bonds that define chemical structure but
with the weak forces that act under relatively mild physical conditions. The structures of most
biological molecules are determined by the collective influence of many individually weak
interactions. The weak electrostatic forces that interest biochemists include ionic interaction,
hydrogen bonds and van der waal forces.

The strength of association of ionic groups of opposite charge depends on the chemical nature of
the ions, the distance between them and the polarity of the medium. In general, the strength of
the interaction between two charged groups (i.e., the energy required to completely separate in
the medium of interest) is less than the energy of a hydrogen bond.

The non-covalent associations between neutral molecules, collectively known as van der -waals
forces, arises from electrostatic interaction among permanent or induced dipoles (the hydrogen
bond is a special kind of dipolar interaction). Interactions among permanent dipoles such as
carbonyl groups are much weaker than ionic interactions. A permanent dipole also induces a
dipole moment in a neighboring group by electrostatically distorting it electron distribution. Such
dipole induced interactions are generally much weaker than dipole-dipole interaction.

Higher orders of protein structures are stabilized primarily- and often exclusively – by
noncovalent interactions. Principal among these are hydrophobic interactions that drive most
hydrophobic amino acids side chains into the interior of the proteins, shielding them from water.
Other significant contributors include hydrogen bonds and salt bridges between the carboxylate
of aspartate

Water is an excellent Solvent

The polar character of water makes it an excellent solvent for polar and ionic materials, which
are said to be hydrophilic. On the other hand, nonpolar substances are virtually insoluble in water
and are consequently described as hydrophobic. Water is an excellent solvent for both ionic
compound (e.g., NaCl) and low molecular weight nonionic polar compounds (e.g., sugar and
alcohol). Ionic compounds are soluble because water can overcome the electrostatic attraction
between ions through solvation of the ions. Nonionic polar compounds are soluble because water
molecules can form hydrogen bonds to polar groups (e.g., -OH).

Amphipathic compounds which contain both large non polar hydrocarbon chains (hydrophobic
groups) and polar or ionic groups (hydrophilic groups) may associate with each other in

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submicroscopic aggregations called micelles. Micelles have hydrophilic (water liking) groups on
their exterior (bonding with solvent water), and hydrophobic water (water disliking) groups
clustered in their interior. They occur in spherical, cylindrical or ellipsoidal shapes. Micelles
structures are stabilized by hydrogen bonding with water, by van der waals attractive forces
between hydrocarbon groups in the interior and by energy of hydrophobic reactions. As with
hydrogen bonds, each hydrophobic interaction is very weak but many such interactions result in
formation of large stable structures.

Hydrophobic interaction plays a major role in maintaining the structure and functions of cell
membranes, the activity of proteins, the anesthetic action of non-polar compound such as
chloroform and nitrous oxide, the absorption of digested fats and the circulation of hydrophobic
molecules in the interior of micelles in blood plasma.

Polar solvents such as water weaken the attractive forces between oppositely charged ions (such
as Na+ and Cl-) and can therefore hold the ions apart. (In nonpolar solvents, ions of opposite
charge attract each other so strongly that they coalesce to form a solid salt.) An ion immersed in
a polar solvent such water attracts the oppositely charged ends of the solvent dipoles. The ion is
thereby surrounded by one or more concentric shells of oriented solvent molecules. Such ions are
said to be solvated or, when water is the solvent, to be hydrated.

The bond dipoles of uncharged polar molecules make them soluble in aqueous solutions for the
same reasons that ionic substances are water soluble. The solubility‘s of polar and ionic
substances are enhanced when they carry functional groups, such as hydroxyl (OH), carbonyl
(C=O), carboxylate (COO-), or ammonium (NH3+) groups, that can form hydrogen bond with
water. Indeed, water soluble biomolecules such as proteins, nucleic acids and carbohydrates
bristle with just such groups. Nonpolar substances, in contrast, lack hydrogen bonding donor and
acceptor groups.

BONDS IN BIOLOGICAL SYSTEM

3. Nonpolar or Hydrophobic Bond. Many amino acids (like alanine, valine, leucine, isoleucine,
Methionine, tryptophan, phenylalanine and tyrosine) have the side chains or R groups which are
essentially hydrophobic, i.e., they have little attraction for water molecules in comparison to the
strong hydrogen bonding between water molecules. Such R groups can unite among themselves
with elimination of water to form linkages between various segments of a chain or between
different chains. This is very much like the coalescence of oil droplets suspended in water. The
association of various R groups in this manner leads to a relatively strong bonding. It also serves
to bring together groups that can form hydrogen bonds or ionic bonds in the absence of water.
Each type linkage, thus, helps in the formation of the other; the hydrophobic bonds being most
efficient in this aspect. The hydrophobic bonds also play important role in other protein

6
interactions, for example, the formation of enzyme-substrate complexes and antibody- antigen
interactions.

1. Disulfide Bond (-S-S-). In addition to the peptide bond, a second type of covalent bond found
between amino acid residues in proteins and polypeptides is the disulfide bond, which is formed
by the oxidation of the thiol or sulfhydryl (-SH) groups of two cysteine residues to yield a mole
of cystine, an amino acid with a disulfide bridge . In generalized form, the above reaction may be
written as:

Insulin is another excellent example where two peptide chains are linked together by 2 disulfide bonds.
The presence of an internal disulfide bond in the glycyl (or A) chain between residues 6 and 11 is
noteworthy.

4. Ionic or Electrostatic Bond or Salt linkage or Salt Bridge. Ions possessing similar charge repel each
other whereas the ions having dissimilar charge attract each other. For example, divalent cations like
magnesium may form electrostatic bonds with 2 acidic side chains. Another instance of ionic bonding
may be the interaction between the acidic and basic groups of the constituent amino acids shown at the
bottom of Fig. 9−13. The R groups of glutamic acid and aspartic acid contain negatively charged
carboxylate groups, and the basic amino acids (arginine, histidine, and lysine) contain positively charged
amino groups in the physiological pH range. Thus, these amino acids contribute negatively charged and
positively charged side chains to the polypeptide backbone. When two oppositely charged groups are
brought close together, electrostatic interactions lead to a strong attraction, resulting in the formation
of an electrostatic bond. In a long polypeptide chain containing a large number of charged side chains,
there are many opportunities for electrostatic interaction. Intramolecular ionic bonds are rather
infrequently used in the stabilization of protein structure but when they are so used, it is often with
great effect. In fact, ionized groups are more frequently found stabilizing interactions between protein
and other molecules. Thus, ionic bonds between positively charged groups (side chains of lysine,
arginine and histidine) and negatively charged groups (COO − group of side chain of aspartic and
glutamic acids) do occur. These ionic bonds, although weaker than the hydrogen bonds, are regarded as
responsible for maintaining the folded structure (or the tertiary structure) of the globular proteins.

2. Hydrogen Bond (>CO......HN<). When a group containing a hydrogen atom, that is

covalently-bonded to an electronegative atom, such as oxygen or nitrogen, is in the vicinity of a
second group containing an electronegative atom, an energetically favourable interaction occurs
which is referred to as a hydrogen bond. The formation of a hydrogen bond is due to the
tendency of hydrogen atom to share electrons with two neighbouring atoms, esp., O and N. For
example, the carbonyl oxygen of one peptide bond shares its electrons with the hydrogen atom of
another peptide bond. Thus,

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THE HYDROPHOBIC EFFECT IN WATER

When a nonpolar substance is added to an aqueous solution, it does not dissolve but instead is
excluded by the water. The tendency of water to minimize its contact with hydrophobic
molecules is termed the hydrophobic effect. Many large molecules and molecular aggregates,
such as proteins, nucleic acids, and cellular membranes, assume their shapes at least partially in
response to hydrophobic effect.

Consider the thermodynamics of transferring a nonpolar molecule from an aqueous solution to a

nonpolar solvent. In all cases, the free energy change is negative, which indicates that such
transfers are spontaneous processes. Interestingly, these transfer processes are either endothermic
(positive ∆H) or isothermic (∆H= 0); that is, it is enthalpically more or less equally favorable for
nonpolar molecules to dissolve in water as in nonpolar media. In contrast, the entropy change
(expressed as -T∆S) is large and negative in all cases. Clearly, the transfer of a hydrocarbon from
an aqueous medium to a nonpolar medium is entropically driven (i.e., the free energy change is
mostly due to an entropy change).

Entropy, or ―randomness‖ is a measure of the order of a system. If entropy increases when a

nonpolar molecule leaves an aqueous solution, entropy must decrease when the molecule enters
water. This decrease in entropy when a nonpolar molecule is solvated by water is an
experimental observation, not a theoretical conclusion. Yet the entropy changes are too large to
reflect only the changes in the conformations of the hydrocarbons. Thus the entropy changes
must arise mainly from some sort of ordering of the water itself.

The hydrophobic effect, which causes nonpolar substances to minimize their contacts with water,
is the major determinant of native protein structure. The aggregation of nonpolar side chains in
the molecules that would otherwise form ordered ―cages‖ around the hydrophobic groups.
Aggregation of the nonpolar molecules increases the entropy of the system, since the number of
water molecules required to hydrate the aggregated solute is less the number of water molecule
required to hydrate the dispersed solute molecules. This increase in entropy accounts for the
spontaneous aggregation of nonpolar substances in water.

Colligative properties

The colligative properties of a solvent depend on the concentration of solute particles. These
properties include freezing point depression, vapor pressure depression, osmotic pressure and
boiling point elevation. The freezing point of
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Osmotic pressure is a measure of the tendency of water molecule to migrate from a dilute to a
concentrated solution through a semipermeable membrane. The migration of water molecules to
is termed osmosis

WATER MOVES BY OSMOSIS AND SOLUTES MOVE BY DIFFUSION

The fluid inside cells and surrounding cells in multicellular organisms is full of dissolved
substances ranging from small inorganic ions to huge molecular aggregates. The concentrations
of these solutes affect water‘s colligative properties, the physical properties that depend on the
concentration of dissolved substances rather than on their chemical features.

Osmosis occurs when two solutions of different concentrations are separated by a membrane
which will selectively allow some species through it but not others. Then, material flows from
the less concentrated to the more concentrated side of the membrane. A membrane which is
selective in the way just described is said to be semipermeable. Osmosis is of particular
importance in living organisms, since most living tissue is semipermeable in one way or another.

In biological systems, the semi permeability relies on a set of solute transporters and channels.
The cell membrane is formed of a lipid bilayer with polar head groups facing out, and nonpolar
hydrocarbon tails in the middle of the membrane. The consequence is that charged and polar
substances cannot cross the membrane. In general, these membranes are impermeable to large
and polar molecules, such as ions, proteins, and polysaccharides, while being permeable to non-
polar and/or hydrophobic molecules like lipids as well as to small molecules like oxygen, carbon
dioxide, nitrogen, nitric oxide, etc. Permeability depends on solubility, charge, or chemistry, as
well as solute size. Water molecules travel through the plasma membrane, tonoplast membrane
(vacuole) or protoplast by diffusing across the phospholipid bilayer via aquaporins (small trans
membrane proteins similar to those in facilitated diffusion and in creating ion channels). Osmosis
provides the primary means by which water is transported into and out of cells. The turgor
pressure of a cell is largely maintained by osmosis, across the cell membrane, between the cell
interior and its relatively hypotonic environment.

Aquaporins are membrane proteins which allow water, but no other molecule, not even H3O+ to
pass through. For other solutes and ions, there exist specific transporters, some which allow a
solute to diffuse down a natural gradient, and others which actively pump ions or other solutes in
or out of the cell. These transporters, pumps and channels can be gated and regulated as well,
allowing a cell to respond to varying osmotic conditions. Osmosis also is the net movement of
solvent molecules through a partially permeable membrane into a region of higher solute
concentration, in order to equalize the solute concentrations on the two sides. It may also be used
to describe a physical process in which any solvent moves, without input of energy, across a
semipermeable membrane (permeable to the solvent, but not the solute) separating two solutions

9
of different concentrations. Although osmosis does not require input of energy, it does use
kinetic energy and can be made to do work.

Osmosis can be explained using the concept of thermodynamic free energy: the less concentrated
solution contains more free energy, so its solvent molecules will tend to diffuse to a place of
lower free energy in order to equalize free energy. Since the semipermeable membrane only
allows solvent molecules to pass through it, the result is a net flow of water to the side with the
more concentrated solution. Assuming the membrane does not break, this net flow will slow and
finally stop as the pressure on the more concentrated side lessens and the movement in each
direction becomes equal: this state is called dynamic equilibrium.

Osmosis can also be explained using the notion of entropy, from statistical mechanics. A system
that has two solutions of different concentrations separated by a semipermeable membrane has
less entropy than a similar system having two solutions of equal concentration. The system with
the differing concentrations is said to be more ordered, and thus has less entropy. The second law
of thermodynamics requires the presence of an osmotic flow that will take the system from an
ordered state of low entropy to a disordered state of higher entropy. Thermodynamic equilibrium
is achieved when the entropy gradient between the two solutions becomes zero.

The tendency for osmotic flow to occur from a solvent to a solution is usually measured in terms
of what is called the osmotic pressure of the solution, symbol Π. This osmotic pressure is not a
pressure which the solution itself exerts but is rather the pressure which must be applied to the
solution (but not the solvent) from outside in order to just prevent osmosis from occurring. The
osmotic pressure is defined to be the pressure required to maintain equilibrium, with no net
movement of solvent. Osmotic pressure is a colligative property, meaning that the osmotic
pressure depends on the molar concentration of the solute but not on its identity.

CHEMICAL PROPERTIES OF WATER

Water is not just a passive component of the cell or extracellular environment. By virtue of its
physical properties, water defines the solubilities of other substances. Similarly, water‘s chemical
properties determine the behavior of other molecules in solution.

IONIZATION OF WATER

As H2O is the medium of biological systems one must consider the role of this molecule in the
dissociation of ions from biological molecules. Water is essentially a neutral molecule but will
ionize to a small degree. This can be described by a simple equilibrium equation:

H2O <——> H+ + OH–

There is actually no such thing as a free proton (H+) in solution. Rather, the proton is associated
with a water molecule as a hydronium ion, H3O+.
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The other product of water‘s ionization is the hydroxide ion, OH-. The proton of a hydronium ion
can jump more rapidly to another water molecule and then to another. Proton jumping is also
responsible for the observation that acid-base reactions are among the fastest reactions that take
place in aqueous solution.

The ionization (dissociation) of water is described by an equilibrium expression in which the

concentration of the parent substance is in the denominator and the concentration of the
dissociated products are in the numerator.

This equilibrium can be calculated as for any reaction:

Because the concentration of the dissociated H2O is so much larger than the concentrations of its
component ions, it can be considered constant and incorporated into K to yield an expression for
the ionization of water,

Kw = [H+][OH–]

The value of Kw, the ionization constant of water is 10-14 at 250C.

This term is referred to as the ion product. In pure water, to which no acids or bases have been
added:

Kw = 1 x 10–14 M2

As Kw is constant, if one considers the case of pure water to which no acids or bases have been
added:

[H+] = [OH-] = 1 x 10–7 M

Pure water must contain equimolar amount of H+ and OH-, Solutions with [H+] = 10-7 M are said
to be neutral, those with [H+]> 10-7 are said to be acidic, and those with [H+] < 10-7 are said to
be basic. Most physiological solutions have hydrogen ion concentrations near neutrality. For
example the human blood is normally slightly basic with [H+] = 4.0 x10-8 M.

A more practical quantity, which was devised in 1909 by Søren Sørenson is known as the pH:

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pH = – log [H+] = log 1/[H+]

The higher the pH, the lower is the H+ concentration; the lower the pH, the higher is the H+
concentration.

Acid-Base Reaction

Johannes Bronsted and Thomas lowry formulated general definition of acid and base, an acid is a
substance that can donate protons and a base is a substance that can accept protons. Under this

HA + H2O <——> H3O+ + A-

A Bronsted acid (HA) react with Bronsted base (H2O) to form the conjugate base of the acid
(A+) and the conjugate acid of the base (H3O+).

HA <——> H+ + A-

Accordingly, the acetate ion (CH3COO-) is the conjugate base of acetic acid (CH3COOH) and
the ammonium ion (NH4+) is the conjugate acid of ammonia (NH3)

Gilbert Lewis described a lewis acid as a substance that can accept an electron pair and a Lewis
base as a substance that can donate an electron pair.

pKa

Acids and bases can be classified as proton donors and proton acceptors, respectively. This
means that the conjugate base of a given acid will carry a net charge that is more negative than
the corresponding acid. In biologically relevant compounds various weak acids and bases are
encountered, e.g. the acidic and basic amino acids, nucleotides, phospholipids etc.

Weak acids and bases in solution do not fully dissociate and, therefore, there is an equilibrium
between the acid and its conjugate base. This equilibrium can be calculated and is termed the
equilibrium constant = Ka. This is also sometimes referred to as the dissociation constant as it
pertains to the dissociation of protons from acids and bases.

In the reaction of a weak acid:

HA <——> A– + H+
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the equilibrium constant can be calculated from the following equation:

As in the case of the ion product:

pKa = –logKa

Therefore, in obtaining the –log of both sides of the equation describing the dissociation of a
weak acid we arrive at the following equation:

Since as indicated above –logKa = pKa and taking into account the laws of logrithms:

From this equation it can be seen that the smaller the pKa value the stronger is the acid. This is
due to the fact that the stronger an acid the more readily it will give up H+ and, therefore, the
value of [HA] in the above equation will be relatively small.

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BUFFERS

A buffer solution is one that resists a change in pH on the addition of acid (H+) or base (OH−),
more effectively than an equal volume of water. Most commonly, the buffer solution consists of
a mixture of a weak Brönsted acid and its conjugate base; for example, mixtures of acetic acid
and sodium acetate or of ammonium hydroxide and ammonium chloride are buffer solutions. A
buffer system consists of a weak acid (the proton donor) and its conjugate base (the proton
acceptor). As an example, a mixture of equal concentrations of acetic acid and acetate ion, found
at the midpoint of the titration curve, is a buffer system. The titration curve of acetic acid has a
relatively flat zone extending about 0.5 pH units on either side of its midpoint pH of 4.76. In this
zone, there is only a small change in pH when increments of either H+ or OH − are added to the
system. This relatively flat zone is the buffering region of the acetic acid-acetate buffer pair. At
the midpoint of the buffering region, where the concentration of the proton donor (acetic acid)
exacts equals that of the proton acceptor (acetate), the buffering power of the system is maximal,
i.e., its pH changes least on addition of an increment of H+ or OH − The pH at this point in the
titration curve of acetic acid is equal to its pKa. The pH of the acetate buffer system does change
slightly when a small amount of H+ or OH− is added, but this change is very small compared
with the pH change that would result if the sameamount of H +(or OH−) were added to pure
water or to a solution of the salt of a strong acid and strong base, such as NaCl, which have no
buffering power. Each conjugate-acid-base pair has a characteristic pH zone in which it is an
effective buffer. The H2PO4−/ HPO42−pair has a pKa of 6.86 and thus can serve as a buffer
system near pH 6.86; the NH4+/NH3 pair, with a pKa of 9.25, can act as a buffer near pH 9.25.

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15
Henderson−Hasselbalch Equation

The quantitative relationship among pH, buffering action of a mixture of weak acid with its
conjugate base, and the pKa of the weak acid is given by a simple expression called Henderson-
Hasselbalch Equation. The titration curves of acetic acid, H2PO4−and NH4+ have nearly identical
shapes, suggesting that they all point towards a fundamental law or relationship. This is actually
the case. The shape of the titration curve of any weak acid is expressed by Henderson−
Hasselbalch equation. This equation is simply a useful way of restating the expression for the
dissociation constant of an acid. For the dissociation of a weak acid HA into H+and A−, the
Henderson−Hasselbalch equation can be derived as follows:

1. Rearrange the Ka equation to solve for (H+] :

2. Convert to logarithmic functions:

3. Make the expression negative (or multiply by −1):

4. Substitute pH for −log [H+] and pKa for −log Ka

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5. Now, to remove the minus sign, invert the last term, i.e., −log [HA]/[A ]−to obtain
HendersonHasselbalch equation :

The equation is expressed more generally as:

This equation fits the titration curve of all weak acids and enables one to deduce a number of
important quantitative relationships. Henderson-Hasselbalch equation is of great predictive value
in protonic equilibria as illustrated below:

A. When [A−] = [HA] or when an acid is exactly half neutralized: Under these conditions,

Therefore, at half neutralization, pH = pKa. The equation, thus, shows why the pKa of a weak
acid is equal to the pH of the solution at the midpoint of its titration.

B. When the ratio [A−]/[HA] = 100 to 1 :

C. When the ratio [A−]/[HA] = 1 to 10 :

If the equation is evaluated at several ratios of [A–] / [HA] between the limits 103 and 10–3, and
the calculated pH values plotted, the result obtained describes the titration curve for a weak acid.

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BIOLOGICAL BUFFER SYSTEMS

Almost every biological process is pH-dependent; a small change in pH produces a large change
in the rate of the process. This is true not only for the many reactions in which the H+ ion is a
direct participant, but also for those in which there is no apparent role for H+ ions. The enzymes
and many of the molecules on which they act, contain ionizable groups with characteristic pK a
values. The protonated amino (−NH3+) and carboxylic groups of amino acids and the phosphate
groups of nucleotides, for example, function as weak acids; their ionic state depends upon the pH
of the solution in which they are dissolved. Cells and organisms maintain a specific and constant
cytosolic pH, keeping biomolecules in their optimal ionic state, usually near pH 7. In multicelled
organisms, the pH of the extracellular fluids (blood, for example) is also tightly regulated.
Constancy of pH is achieved primarily by biological buffers: mixtures of weak acids and their
conjugate bases. The table below shows some important buffering systems of body fluids which
help maintaining pH. A certain amount of many of these is usually present in the body and
cellular fluids, and so the maintenance of a constant pH depends on a complex system.

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1. The Phosphate Buffer System

This system, which acts in the cytoplasm of all cells, consists of H2PO4–as proton donor and
HPO42– as proton acceptor:

The phosphate buffer system works exactly like the acetate buffer system, except for the pH
range in which it functions. The phosphate buffer system is maximally effective at a pH close to
its pKa of 6.86, and thus tends to resist pH changes in the range between 6.4 and 7.4. It is,
therefore, effective in providing buffering power in intracellular fluids. Since the concentration
of phosphate buffer in the blood plasma is about 8% of that of the bicarbonate buffer, its
buffering capacity is much lower than bicarbonate in the plasma. The concentration of phosphate
buffer is much higher in intracellular fluid than in extracellular fluids. The pH of intracellular
fluids (6.0 −6.9) is nearer to the pKa of the phosphate buffer. Therefore, the buffering capacity of
the phosphate buffer is highly elevated inside the cells and the phosphate is also effective in the
urine inside the renal distal tubules and collecting ducts.

2. The Bicarbonate Buffer System

This is the main extracellular buffer system which (also) provides a means for the necessary removal of
the CO2 produced by tissue metabolism. The bicarbonate buffer system is the main buffer in blood
plasma and consists of carbonic acid as proton donor and bicarbonate as proton acceptor:

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The pH of a bicarbonate buffer system depends on the concentration of H2CO3and HCO3−, the proton
donor and acceptor components. The concentration of H2CO3, in turn, depends on the concentration of
dissolved CO2, which, in turn, depends on the concentration or partial pressure of CO2 in the gas phase.
With respect to the bicarbonate system, a [HCO3−] / [H2CO3] ratio of 20 to 1 is required for thepH of
blood plasma to remain 7.40.

3. The Protein Buffer Systems

The protein buffers are very important in the plasma and the intracellular fluids but their
concentration is very low in cerebrospinal fluid, lymph and interstitial fluids. The proteins exist
as anions serving as conjugate bases (Pr−) at the blood pH 7.4 and form conjugate acids (HPr)
accepting H+. They have the capacity to buffer some H2CO3 in the blood.

4. The amino acids buffer system

This system also operates in humans. Amino acids contain in their molecule both an acidic
(−COOH) and a basic (−NH2) group. They can be visualized as existing in the form of a neutral
zwitterion in which a hydrogen atom can pass between the carboxyl and amino groups. The
glycine may, thus, be represented as:

By the addition or subtraction of a hydrogen ion to or from the zwitterion, either the cation or
anion form will be produced:

Thus, when OH− ions are added to the solution of amino acid, they take up H+ from it to form
water, and the anion is produced. If H+ions are added, they are taken up by the zwitterion to
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produce the cation form. In practice, if NaOH is added, the salt H2N-CH2-COONa would be
formed H+ and the addition of HCl would result in the formation of amino acid hydrochloride,
Cl-H -H3N-CH2-COOH, but these substances would ionize in solution to some extent to form
their corresponding ions. Hemoglobin and plasma proteins act as buffers in a similar way.Amino
acids differ in the degree to which they will produce the cation or anion form. In other words, a
solution of an amino acid is not neutral but is either predominantly acidic or basic, depending on
which form is present in greater quantity. For this reason, different amino acids may be used as
buffers for different pH values, and a mixture of them possesses a wide buffer range.

5. The Hemoglobin Buffer Systems

These buffer systems are involved in buffering CO2 inside erythrocytes. The buffering capacity
of hemoglobin depends on its oxygenation and deoxygenation. Inside the erythrocytes, CO2
combines with H2O to form carbonic acid (H2CO3) under the action of carbonic anhydrase. At
the blood pH 7.4, H2CO3dissociates into H+ and HCO3−and needs immediate buffering.
Oxyhemoglobin (HbO2–), on the other side, loses O2to form deoxyhemoglobin (Hb−) which
remains undissociated (HHb) by accepting H+from the ionization of H2CO3. Thus, Hb− buffers
H2CO3 in erythrocytes :

Some of the HCO3− diffuse out into the plasma to maintain the balance between intracellular and
plasma bicarbonates. This causes influx of some Cl− into erythrocytes along the electrical
gradient produced by the HCO3− outflow (chloride shift). HHbO2, produced in lungs by
oxygenation of HHb, immediately ionizes into H+ and HbO2−. The released hydrogen ions (H+)
are buffered by HCO3– inside erythrocyte to form H2CO3 which is dissociated into H2O and CO2
by carbonic anhydrase. CO2 diffuses out of erythrocytes and escapes in the alveolar air. Some
HCO3– return from the plasma to erythrocytes in exchange of Cl− and are changed to CO2 .

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 THERMODYNAMICS

Thermodynamics is the study of energy in systems, and the distribution of energy among
components. In chemical systems, it is the study of chemical potential, reaction potential,
reaction direction, and reaction extent. If we know the energy changes associated with a reaction
or process, we can predict the equilibrium concentrations. We can also predict the direction of a
reaction provided we know the initial concentrations of reactants and products. The
thermodynamic quantity that provides this information is the Gibbs free energy.

FREE ENERGY
The energy actually available to do work (utilizable) is known as free energy. The Gibbs free
energy change (∆G) for a reaction is the difference between the free energy of the products and
of the reactants. The overall Gibbs free energy change has two components known as the
enthalpy change (∆H, the change in heat content) and the entropy change (∆S, the change in
randomness). A biochemical process may generate heat or absorb it from the surroundings.
Similarly, a process may occur with an increase or a decrease in the degree of disorder, or
randomness, of the reactants.

∆G = ∆H - T∆S

Starting with an initial solution of reactants and products, if the reaction proceeds to produce
more products, then ∆G must be less than zero (∆G < 0). In chemistry terms, we say that the
reaction is spontaneous and energy is released. When ∆G is greater than zero (∆G > 0), the
reaction requires external energy to proceed and it will not yield more products. In fact more
reactants will accumulate as the reverse reaction is favored. When ∆G equals zero (∆G = 0), the
reaction is at equilibrium; the rate of the forward and reverse reactions are identical and the
concentrations of the products and reactant no longer change.

A series of linked processes, such as the reactions of a metabolic pathway in a cell, usually
proceeds only when associated with an overall negative Gibbs free energy change. Biochemical
reactions or processes are more likely to occur, both to a greater extent and more rapidly, when
they are associated with an increase in entropy and a decrease in enthalpy.

If we know the Gibbs free energy of every product and every reactant, it would be a simple
matter to calculate the free energy change for a reaction by using:

∆Greaction = ∆Gproducts - ∆Greactants

What we need are some standard values of ∆G that can be adjusted for concentration. These
standards are the Gibbs free energy changes measured under certain conditions. By convention,
standard conditions are 25oC (298K), 1 atm standard pressure, and 1.0 M of all products and
reactants. In most biochemical reaction, the concentration of H+ is important, and this is
22
indicated by the pH. The standard condition for biochemistry reactions is pH = 7.0, which
correspond to 10-7 M H+ (rather than 1.0 M as for other reactants and products). The Gibbs free
energy change under these standard conditions is indicated by the symbol ∆Go'.

The actual Gibbs free energy is related to its standard free energy by

∆GA = ∆GAo' + RT ln [A]

Where R is the universal gas constant (8.315kJ-1) and T is the temperature in kelvin. The term
RTln[A] is sometimes given as 2.303 RTlog[A].

CHEMICAL KINETICS

Kinetic experiments examine the relationship between the amount of product (P) formed in a unit
time (∆[p]/∆t) and the experimental conditions under which the reaction take place. The basis of
most kinetic measurements is the observation that the rate, or velocity (v), of a reaction varies
directly with the concentration of each reactant. This observation is expressed in a rate equation.
For example the rate equation for the non- enzymatic conversion of substrate (S) to product in an
isomerization reaction is written as

∆[p]/∆t = v = k[s]

The rate equation reflects the fact that the velocity of a reaction depends on the concentration of
the substrate ([s]). The symbol k is the rate constant and indicates the speed or efficiency of a
reaction. Each reaction has a different rate constant. The units of the rate constant for a simple
reaction are s-1..

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As a reaction proceeds, the amount of product ([p]) increases and the amount of substrate ([s])
decreases. An example of the progress of several reactions is shown in graph above. The velocity
is the slope of the progress curve over a particular interval of time. The shape of the curves
indicates that the velocity is decreasing over time as expected since the substrate is being
depleted.

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OXIDATION-REDUCTION REACTIONS

Oxidation-reduction reactions are central to the supply of biological energy. In an oxidation-

reduction (redox) reaction, electrons from one molecule are transferred to another. Oxidation is
the loss of electrons: a substance that is oxidized will have fewer electrons when the reaction is
complete. Reduction is the gain of electrons: a substance that gains electrons in a reaction is
reduced. Oxidation and reduction reactions always occur together. One substrate is oxidized and
the other is reduced. An oxidizing agent is a substance that causes an oxidation-it takes electrons
from the substrate that it oxidized. Thus, oxidizing agents gain electrons (i.e., they are reduced).
A reducing agent is a substance that donates electrons (and is oxidized in the process).

Oxidation can take several forms, such as removal of hydrogen (dehydrogenation), addition of
oxygen, or removal of electrons. Dehydrogenation is the most common form of biological
oxidation. Oxidoreductases (enzymes that catalyze oxidation-reduction reactions) represent a
large class of enzymes and dehydrogenases (enzymes that catalyze removal of hydrogen) are a
major subclass of oxidoreductases.

Most dehydrogenations occur by C-H bond cleavage producing a hydride ion (H- ). The substrate
is oxidized because it loses the electrons associated with the hydride ion. Such reactions will be
accompanied by a corresponding reduction where another substrate gains electron by reacting
with the hydride ion. The dehydrogenation of lactate is an example of removal of hydrogen. In
this case the oxidation of lactate is coupled to the reduction of the coenzyme NAD+.

The nicotinamide coenzymes play a role in many oxidation-reduction reactions. They assist in
the transfer of electrons to and from metabolite. The oxidized forms, NAD+ and NADP+, are
electron deficient and the reduced forms, NADH and NADPH carry an extra pair electron in the
form of a covalently bound hydride ion.

Electrochemical cell

Test

1. Describe the hydrogen bonding in water

This is a chemical bond between hydrogen and an electronegative atom such as oxygen, chlorine
etc. The attraction between one of the slightest positive hydrogen atoms of one water molecule
and the slightest negative electron pairs in one of the sp3 hybrid orbitals produces a hydrogen
bond. In a hydrogen bond between two water molecules, the hydrogen atom remains covalently
bonded to its oxygen atom, the hydrogen donor. At the same time, it is attracted to another
oxygen atom called the hydrogen acceptor. In effect, the hydrogen atom is being shared
(unequally) between the two oxygen atoms. The distance from the hydrogen atom to the acceptor
oxygen is about twice the length of the covalent bond. The distance from the hydrogen atom to
25
the acceptor oxygen is about twice the length of the covalent bond. Water is the only molecule
capable of forming hydrogen bonds; these interactions can occur between any electronegative
atom and hydrogen attached to another electronegative atom. Hydrogen bonds are much weaker
than typical covalent bonds. The strength of hydrogen bonds in water and in solution is estimated
to be about 20 kJ mol‾¹. The strength of hydrogen bond is less than 5% of the strength of typical
covalent bonds. Hydrogen bonds are weak interactions compared to covalent bonds but their
large number is the reason for the stability of liquid water. Water molecules are unusual because
they can form four O—H—O aligned hydrogen bonds with up to four other water molecules.
They can donate each of their two hydrogen atoms to two other water molecules and accept two
hydrogen atoms from two other water molecules. Each hydrogen atom can participate in only
one hydrogen bond.

H—O—H + H—O—H → O—H-----O ∆Hƒ -20 kJ mol‾¹

Hydrogen Bonding

2. What are osmosis and its advantages in a biological system?

Osmosis occurs when two solutions of different concentrations are separated by a membrane
which will selectively allow some species through it but not others. Then, material flows from
the less concentrated to the more concentrated side of the membrane. A membrane which is
selective in the way just described is said to be semipermeable. Osmosis is of particular
importance in living organisms, since most living tissue is semipermeable in one way or another.
Osmosis also is the net movement of solvent molecules through a partially permeable membrane
into a region of higher solute concentration, in order to equalize the solute concentrations on the
two sides. It may also be used to describe a physical process in which any solvent moves,
without input of energy, across a semipermeable membrane (permeable to the solvent, but not
the solute) separating two solutions of different concentrations. Although osmosis does not
require input of energy, it does use kinetic energy and can be made to do work.

The tendency for osmotic flow to occur from a solvent to a solution is usually measured in terms
of what is called the osmotic pressure of the solution, symbol Π. This osmotic pressure is not a
pressure which the solution itself exerts but is rather the pressure which must be applied to the
solution (but not the solvent) from outside in order to just prevent osmosis from occurring. The
osmotic pressure is defined to be the pressure required to maintain equilibrium, with no net
movement of solvent. Osmotic pressure is a colligative property, meaning that the osmotic
pressure depends on the molar concentration of the solute but not on its identity.

26
 3. Using Henderson Hasselbalch equation describe buffering and its advantage in a
biological system.

Henderson-Hasselbalch equation: the relationship between the pH of a solution and the

concentrations of an acid and its conjugate base by rearranging:

[H+] = K([H+]/[A+]

Taking log of both side of the equation (above)

It should be obvious now that the pH of a solution of any acid (for which the equilibrium
constant is known, and there are numerous tables with this information) can be calculated
knowing the concentration of the acid, HA, and its conjugate base [A–]. This equation
indicates that the pK of an acid is numerically equal to the pH of the solution when the
molar concentrations of the acid and its conjugate base are equal.

At the point of the dissociation where the concentration of the conjugate base [A–] = to
that of the acid [HA]:

pH = pKa + log[1]

The log of 1 = 0, Thus, at the mid-point of a titration of a weak acid:

pKa = pH

In other words, the term pKa is that pH at which an equivalent distribution of acid and
conjugate base (or base and conjugate acid) exists in solution. The Henderson-
Hasselbalch equation is often used by chemists and biologists to calculate the pH of a
buffer.

4. What is hydrophobic effect?

When a nonpolar substance is added to an aqueous solution, it does not dissolve but instead is
excluded by the water. The tendency of water to minimize its contact with hydrophobic
molecules is termed the hydrophobic effect. Many large molecules and molecular aggregates,
such as proteins, nucleic acids, and cellular membranes, assume their shapes at least partially in
response to hydrophobic effect. The hydrophobic effect, which causes nonpolar substances to
minimize their contacts with water, is the major determinant of native protein structure. The
aggregation of nonpolar side chains in the molecules that would otherwise form ordered ―cages‖
around the hydrophobic groups. Aggregation of the nonpolar molecules increases the entropy of
27
the system, since the number of water molecules required to hydrate the aggregated solute is less
the number of water molecule required to hydrate the dispersed solute molecules. This increase
in entropy accounts for the spontaneous aggregation of nonpolar substances in water.

5. What is pH?

pH = – log [H+] = log 1/[H+]

The higher the pH, the lower is the H+ concentration; the lower the pH, the higher is the H+
concentration.

[H+] = [OH-] = 1 x 10–7 M

Pure water must contain equimolar amount of H+ and OH-, Solutions with [H+] = 10-7 M are said
to be neutral, those with [H+]> 10-7 are said to be acidic, and those with [H+] < 10-7 are said to
be basic. Most physiological solutions have hydrogen ion concentrations near neutrality. For
example the human blood is normally slightly basic with [H+] = 4.0 x10-8 M.

3. Buffer is a solution that resist the change in pH as result of change in hydrogen or hydroxyl
ion concentration. The ability of a buffer to resist pH changes with added acid or base is directly
proportional to the total concentration of the conjugate acid-base pair, [HA] + [A-]. It is maximal
when pH = pK and decreases rapidly with a change in pH from that point. When [HA] = [A-],
the pH of the solution is relatively insensitive to the addition of strong base or strong acid. Such
a solution, which is known as an acid-base buffer, is resistant to pH changes because small
amounts of added H+ or OH-, respectively, react with A- or HA present without greatly changing
the value of log ([A-]/[HA]).

4. Water is a ‗universal solvent‘

The polar character of water makes it an excellent solvent for polar and ionic materials, which
are said to be hydrophilic. On the other hand, nonpolar substances are virtually insoluble in water
and are consequently described as hydrophobic. Polar solvents such as water weaken the
attractive forces between oppositely charged ions (such as Na+ and Cl-) and can therefore hold
the ions apart. (In nonpolar solvents, ions of opposite charge attract each other so strongly that
they coalesce to form a solid salt.) An ion immersed in a polar solvent such water attracts the
oppositely charged ends of the solvent dipoles. The ion is thereby surrounded by one or more
concentric shells of oriented solvent molecules. Such ions are said to be solvated or, when water
is the solvent, to be hydrated.

6b. water is not the only polar solvent. Oxygen atoms are more electronegative than hydrogen
atoms because an oxygen nucleus attracts electron attracts electrons more strongly than the
single proton in the hydrogen nucleus. As a result, an uneven distribution of charge occurs within
each O-H bond of the water molecule with oxygen bearing a partial negative charge and
28
hydrogen bearing a partial positive charge. This uneven distribution of charge within a bond is
known as a dipole and the bond is said to be polar. The polarity of a molecule depends on the
polarity of its covalent bonds and it geometry. The angled arrangement of the polar O-H bonds
of water creates a permanent dipole for the molecule. Water and gaseous ammonia are
electrically neutral, both molecule are polar. The high solubility of polar ammonia molecules in
water is facilitated by strong interactions with polar water molecules. The solubility of ammonia
in water demonstrates the principle that ―likes dissolves likes‖.

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