BIOCHEMISTRY (BICHEM)

I. CHAPTER 2: WATER

II. LESSON TITLE

1. Physical and Chemical Properties of Water

III. LESSON OVERVIEW

Any study of the chemistry of life must include a study of water. Water is the
most abundant substance in Living systems, making up 70% of the weight of most
organisms. The first living organisms on Earth doubtless arose in an aqueous
environment, and the course of evolution has been shaped by the properties of
the aqueous medium in which life began.
This chapter begins with a description of the physical and chemical properties
of water, to which all aspects of cell structure and function are adapted. The
attractive forces between water molecules and the slight tendency of water to
ionize are of crucial importance to the structure and function of biomolecules. The
water molecule and its ionization products, H+ and OH-, profoundly influence the
structure, self-assembly, and properties of all cellular components, including
proteins, nucleic acids, and lipids. The non-covalent interactions responsible for
the strength and specificity of recognition among biomolecules are decisively
influenced by the solvent properties of water, including its ability to form hydrogen
bonds with itself and with solutes.
IV.DESIRED LEARNING OUTCOMES

At the end of the chapter, students should be able to:

1. Explain the polarity of the water molecule

2. Compare the structure of water in a solid and in a liquid state
3. Solve problems with pH, pOH, and buffers.
4. Discuss the importance of maintaining biological molecules in a buffer solution

V. LESSON CONTENT

The colorless, odorless, and tasteless nature of water belies its fundamental
importance to living organisms. Despite its bland appearance to our senses, water is
anything but inert. Its physical properties-unique among molecules of similar size-
give its unparalleled strength as a solvent. And yet its limitations as a solvent also
have important implications for the structure and functions of biological molecules.

A. Physical Properties of Water

Water is a Polar Molecule

A water molecule consists of two hydrogen atoms bonded to an oxygen atom.

The O-H bond distance is 0.958 A0 (1A0= 10-10 m), and the angle formed by the three
atoms is 104.5O. The hydrogen atoms are not arranged linearly, because the oxygen
atom’s four sp3 hybrid orbitals extend roughly toward the corners of the tetrahedron.
Hydrogen atoms occupy a tetrahedron, and the non-bonding electron pairs of the

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oxygen atom occupy the other two corners (in a perfectly tetrahedral molecule, such
as methane, and CH4) the bond angles are 109.50.

a) The shaded outline represents the Van der b) The oxygen atom’s sp3 orbitals are
Waals envelope, the effective “surface” of the arranged tetrahedrally. Two orbitals
molecule. contain nonbonding electron pairs

Figure 2.1. Structure of the Water Molecule

Water Molecules Form Hydrogen Bonds

The angular geometry of the water molecule has enormous implications for
living systems. Water is a polar molecule: the oxygen atom with its unshared
electrons carries a partial negative charge (δ -), and hydrogen atoms each carry a
partial positive charge (δ+). Electrostatic attractions between the dipoles of water
molecules are crucial to the properties of water itself and its role as a biochemical
solvent. Neighboring water molecules tend to orient themselves so that the O-H bond
of one water molecule (the positive end) points toward one of the electron pairs of the
other water molecule (the negative end). The resulting directional intermolecular
association is known as a hydrogen bond.

Figure 2.2 A hydrogen bond

between two water molecules. The
strength of the interaction is
maximal when the O-H covalent
bond of one molecule points directly
toward to the lone-pair electron
cloud of the other.

In general, a hydrogen bond can be represented as D—H ···A, where D—H

is a weakly acidic “donor” group such as O—H, N—H, or sometimes S—H and A is a
weakly basic “acceptor” atom such as O, N, or occasionally S.
A single water molecule contains two hydrogen atoms that can be “donated
and two unshared electron pairs that can act as “acceptors, so each molecule can
participate in a maximum of four hydrogen bonds with other water molecules.

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Figure 2.3 Hydrogen bonding in ice. In

ice, each water molecule forms four
hydrogen bonds, the maximum possible for
a water molecule, creating a regular crystal
lattice. BY contrast, in liquid water at room
temperature and atmospheric pressure,
each water molecule hydrogen-bonds with
an average of 3.4 other water molecules.
This crystal lattice structure makes ice less
dense than liquid water, and thus ice floats
on liquid water.

Hydrogen Bonds and other Weak Interactions Influence Biological Molecules

The structures of most biological molecules are determined by the collective
influence of many individual weak interactions. The weak electrostatic forces that
interest biochemists include ionic interactions, hydrogen bonds, and Van der Waals
forces.

Type of Bond Example Bond Strength

(kJ.mol-1)
O—H 460
Covalent C—H 414
C—C 348
Non-covalent
Ionic interaction --COO- +
H3N-- 86
Van der Waals forces
20
Hydrogen bond
Dipole-dipole interaction 9.3

London dispersion forces 0.3

Figure 2.4. Bond Energies in Biomolecules

The non-covalent association between neutral molecules, collectively known

as Van der Waals forces, arises from electrostatic interactions among permanent or
induced dipoles (hydrogen bond is a special kind of dipolar interaction). London
dispersion forces are extremely weak and fall off so rapidly with the distance that
they are significant only for the group in close contact. They are nevertheless,
extremely important in determining the structures of biological molecules, whose
interiors contain many closely packed groups.

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A) Interactions between B) Dipole-induced dipole C)London dispersion
permanent dipoles interactions forces

Figure 2.5 Dipole-dipole interactions. The strength of each dipole is indicated by the thickness of
the accompanying arrow.

Hydrophilic Substances Dissolve in Water

Solubility depends on the ability of a solvent to interact with a solute more
strongly than solute particles interact with each other. Water is said to be the
universal solvent. The polar character of water makes it an excellent solvent for polar
and ionic materials, which are said to be hydrophilic (Greek: hydro, water+ philos,
loving). On the other hand, non-polar substances are virtually insoluble in water (“oil
and water don’t mix”) and are consequently described as hydrophobic (Greek:
phobos, fear). Non-polar substances, however, are soluble in nonpolar solvents such
as carbon tetrachloride (CCl4) and hexane (CH4). This information is summarized by
another maxim, “like dissolves like”.
Why do salts such as sodium chloride (NaCl) dissolve in water? Polar
solvents, such as water, weaken the attractive forces between oppositely charges
ions (such as Na+ and Cl-) and can therefore hold the ions part. (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 as 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.

Figure 2.6 Solvation of ions. The dipoles of the surrounding water molecules
are oriented according to the charge of the ion. Only one layer of solvent
molecules is shown.

The energetics of solvation also play a role in chemical reactions, since a reacting
group must shed its water hydration (the molecules in its hydration shell) to closely
approach another group.

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Between a hydroxyl group of Between a carbonyl group of Between an amino group of

alcohol and H2O a ketone and H2O an amine and H2O
Figure 2.7 Hydrogen bonding between polar groups and water

Bonding Arrangement Molecules Where the Bond Occurs

H bond found in H2O

Bonding of H2O to other molecules

Important in protein and nucleic acid structures

Figure 2.8 Examples of Major Types of Hydrogen Bonds Found in Biologically Important
Molecules

The Hydrophobic Effect Causes Non-polar Substances to Aggregate in Water

When a non-polar 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 as hydrophobic effect. Many large
molecules and molecular aggregates, such as proteins, nucleic acids, and cellular
membranes, assume their space at least in response to the hydrophobic effect.
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 extensive hydrogen-bonding network of liquid water molecules is
disrupted when a nonpolar group intrudes. A nonpolar group can neither accept nor
donate hydrogen bonds, so the water molecules at the surface of the cavity occupied
by the nonpolar group cannot hydrogen bond to other molecules in their usual
fashion. In order to maximize their hydrogen-bonding ability, these surface water
molecules orient themselves to form a hydrogen-bonded network enclosing the cavity
(Fig. 2-9). This orientation constitutes an ordering of the water structure since the number
of ways that water molecules can form hydrogen bonds around the surface of a nonpolar
group is fewer than the number of ways, they can form hydrogen bonds in bulk water.

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Unfortunately, the ever-fluctuating nature of liquid water’s basic structure has
not yet allowed a detailed description of this ordering process. One model proposes
that water forms icelike hydrogen-bonded “cages” around the nonpolar groups. The
water molecules of the cages are tetrahedrally hydrogen bonded to other water
molecules, and the ordering of water molecules extends several layers beyond the
first hydration shell of the nonpolar solute.
The unfavorable free energy of hydration of a nonpolar substance caused by
its ordering of the surrounding water molecules has the net result that the nonpolar
substance tends to be excluded from the aqueous phase. This is because the
surface area of a cavity containing an aggregate of nonpolar molecules is less than
the sum of the surface areas of the cavities that each of these molecules would
individually occupy (Fig. 2-10). The aggregation of the nonpolar groups thereby
minimizes the surface area of the cavity and therefore maximizes the entropy of the
entire system. In a sense, the nonpolar groups are squeezed out of the aqueous
phase

Figure 2.9 Orientation of Figure 2.10. Aggregation of non-polar molecules in water.

water molecules around a
non-polar solute. To maximize
their number of hydrogen
bonds, water molecules form a
“cage” around the solute.
Dashed lines represent
hydrogen bonds.
a) The individual hydration of dispersed non-polar molecules
(brown) decreases the entropy of the system because their
hydrating water molecules (dark blue) are not as free to form
hydrogen bonds.
b) Aggregation of the non-polar molecules increases the entropy
of the system since the number of water molecules required to
hydrate the aggregated solutes is less than the number of water
molecules required to hydrate the dispersed solute molecules.
This increase in entropy accounts for the spontaneous
aggregation of non-polar substances in water.

Water Moves by Osmosis and Solute Move by Diffusion

Water movement across a semipermeable membrane driven by differences in
osmotic pressure is an important factor in the life of most cells. Plasma membranes
are more permeable to water than to most other small molecules, ions, and
macromolecules. This permeability is due largely to protein channels in the
membrane that selectively permit the passage of water. Solutions of osmolarity equal
to that of a cell’s cytosol are said to be isotonic relative to that cell. Surrounded by
an isotonic solution, the cell neither gains nor loses water. In a hypertonic solution,
one with higher osmolarity than that of the cytosol, the cell shrinks as water moves
out. In a hypotonic solution, one with a lower osmolarity than the cytosol, the cell
swells as water enters. In their natural environments cells generally contain higher
concentrations of biomolecules and ions than their surroundings, so osmotic
pressure tends to drive water into cells.

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(c) Cell is hypotonic solution;

(b) Cell in hypertonic water moves in creating
solution; water moves out outward pressure: cells swells,
and the cell shrinks may eventually burst

Figure 2.11 Effect of extracellular osmolarity on water movement across plasma membrane.
When a cell in osmotic balance with its surrounding medium-that is, a cell in (a) an isotonic
medium-is transferred into (b) a hypertonic solution or (c) a hypotonic solution, water moves the
plasma membrane in the direction that needs to equalize osmolarity outside and inside the cell.

Diffusion of solutes is the basis for the laboratory techniques of dialysis. In

this process, solutes smaller than the pore size of the dialysis membrane freely
exchange between the sample and the bulk solution until equilibrium is reached.
Large substances cannot cross the membrane and remain where they are. Dialysis is
particularly useful for separating large molecules such as proteins or nucleic acids,
from smaller molecules. Because small solutes (and water) move freely between the
sample and the surrounding medium. Dialysis can be repeated times to replace the
sample medium with another solution.
B. Chemical Properties of Water
Water is not just a passive component of the cell or extracellular environment.
By its physical properties, water defines the solubilities of other substances. Similarly,
water’s chemical properties determine the behavior of other molecules in solution.
Water Ionizes to Form H+ and OH-
Water is a neutral molecule with a very slight tendency to ionize. We express
this ionization as
H2O ⇆ H+ + OH-
Although we commonly show the ionization product of water as H+, free
protons do not exist in solution, hydrogen ions formed in water are immediately
hydrated to hydronium ions (H3O+). Hydrogen bonding between water molecules
makes the hydration of ionizing protons virtually instantaneous.

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The proton of a hydronium ion can jump rapidly to another water molecule
and then to another (Fig 2.12). For this reason, the mobilities of H + and OH- in
solutions are much higher than for other ions, which must move through the bulk
water carrying their waters of hydration. Proton jumping/hopping is also
responsible for the observation that acid-base reactions are among the fastest
reactions that take place in an aqueous solution.

Figure 2.12 Proton jumping. Proton

jumps occur rapidly than direct
molecular migrations, accounting for the
observed high ionic mobilities of
hydronium ions (and hydroxide ions) in
aqueous solutions.

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

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

K is the ionization constant for water. Quantities in square brackets symbolize

the molar concentration of the indicated substances (M=mol/L). Because the
concentration of the unionized H2O is so much larger than the concentration of its
component ions, it can be considered constant and incorporated into K to yield an
expression for the ionization of water.

Kw = ionization constant of water is 1x10-14 (10-14) at 250C.

Pure water must contain equimolar amounts of H+ and OH-, so [H+] = [OH-] = (Kw)1/2 =
10-7 M.

Since [H+] = 1x10-7 (10-7); [OH-] = 1x10-7 (10-7), therefore solutions with

[H+] > 1x10-7 M are said to be acidic: pH < 7

[H+] = 1x10-7 M are said to be neutral; pH = 7
[H+] < 1x10-7 M are said to be basic: pH > 7

Most physiological solutions have hydrogen ion concentrations near neutrality.

For example, human blood is normally slightly basic with [H+] = 4.0 x10-8 M.

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The values of [H+] for most solutions are inconveniently small and thus
impractical to compare. A more practical quantity, which was derived in 1909 by
Soren Sorenson, is known as pH;

The higher the pH, the lower the H+ concentration; the lower the pH, the higher the H+
concentration.
Relationship Between pH and pOH

Kw = [H+] [OH-] = 1.0 x 10-14

pKw = pH + pOH
14= pH + pOH

Example: Calculation of [H+] and pH Example: Calculation of [OH-], pOH, and

pH
1. What is the concentration of H + and
pH in a solution of 0.1M NaOH? 2. What is the concentration of [OH-] in a
solution with a [H+] concentration of 1.3x10-4
Solution: Begin with the equation for the M?
ion product of water.
Solution: Begin with the equation for the ion
+ -
Kw = [H ] [OH ] product of water.

With [OH-] = 0.1 M, solving for [H+] gives Kw = [H+] [OH-]

With [H+]= 1.3x10-4 M, solving for [OH-] gives

pH = - log of [H+]
pH= - log (1x10-13)
pH= 13.0, basic solution
pOH = -log [OH-]
pOH = - log (7.7 x10-11)
pOH = 10.1

14 = pH + pOH
pH = 14 -10.1
pH= 3.9, acidic solution

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Figure 2.13 The pH of some

common aqueous fluids

Acids and Bases alter the pH

H+ and OH- ions derives from water are fundamental to the biochemical
reactions. Biological molecules, such as proteins and nucleic acids, have numerous
functional groups that act as acids or bases, for example, carbonyl and amino
groups. These molecules influence the pH of the surrounding aqueous medium, and
their structures and reactivities are in turn influenced by ambient pH.
An acid can donate a proton. According to a definition formulated in 1923 by
Johannes Bronsted and Thomas Lowry, an acid is a substance that can donate a
proton, and a base is a substance that can accept a proton. Under the Bronsted-
Lowry definition, an acid-base reaction can be written as
HA + H2O ⇄ H3O+ + A-
acid base conjugate acid conjugate base
to H 2O to HA

An acid (HA) reacts with a base (H 2O) to form the conjugate base of the acid (A -) and
the conjugate acid of the base (H3O+).

The acid-base reaction is frequently abbreviated

HA ⇄ H+ + A-

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An alternative expression for basic substance B is

HB ⇄ H+ + B -

The Strength of an Acid is Specified by its Dissociation Constant. The

equilibrium constant of an acid-base reaction is expressed as a dissociation constant
with the concentration of the reactants in the denominator and the concentration of
the products in the numerator:

In dilute solutions, the water concentration is essentially constant, 55.5 M (1000

g.L-1/18.015 g.mol-1 = 55.5 M. Therefore, the term [H 2O] is customarily combined with
the dissociation constant, which can then take form

Because acid dissociation constants, like [H +] values they transformed to pKa values
by the formula
pKa = - log Ka

Acids can be classified according to their relative strengths, that is, their
abilities to transfer a proton to water. Many inorganic acids, such as HClO 4, HNO3,
and HCl, are strong acids. Since strong acids rapidly transfer all their protons to H 2O,
the strongest acid that can exist in aqueous solutions is H3O+. Likewise, there can be
no stronger base in aqueous solutions than OH-. All the acid-base reactions that
occur in biological systems involve H3O+ (and OH-) and weak acids (and their
conjugate bases).

The pH of a Solution is Determined by the Relative Concentrations of Acids

and Bases. The relationship between the pH of a solution and the concentrations of
an acid and its conjugate base is easily derived. The equation can be rearranged to

[H+] = Ka
[ HA ]
¿¿
Taking the negative logarithm of each term gives
pH = - log Ka + log ¿ ¿
Substituting pKa for –log Ka yields

pH = pKa + log ¿ ¿ This relationship is known as the

Henderson-Hasselbalch equation.

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Table 1. Dissociation Constants of Some Acids at 25 OC

Example Problem:
1. Calculate the pH of a 2 L solution containing 10 mL of 5 M acetic acid and 10 ml
of 1 M sodium acetate.
Solution: First, calculate the concentrations of the acid and conjugate base,
expressing all concentrations in units of moles per liter.
Acetic Acid (CH3COOH): ( 0.01 L) (5M)/ (2L) = 0.025 M
Sodium Acetate (NaCH3COO): (0.01) (1M)/ (2L) = 0.005 M

Substitute the concentration of the acid and conjugate base into the Henderson-
Hasselbalch equation, Ka for acetic acid 1.74 x10-5; pKa = 4.76
pH = pKa + log ¿ ¿
pH = pKa + log ([acetate]/[acetic acid])
pH = 4.76 + log (0.005/0.025)
pH = 4.76 - 0.70 = 4.06

Buffer Resist Changes in pH

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 particular but also for those in which there is
no apparent role for the H+ ion.
Cells and organisms maintain a specific and constant cytosolic pH, usually
near pH 7, keeping biomolecules in their optimal ionic state. In multicellular

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organisms, the pH of extracellular fluids is also tightly regulated. The constancy of pH
is achieved primarily by biological buffers: mixtures of weak acids and their conjugate
base.
A buffer is something that resists change. In terms of acid and base chemistry, a
buffer solution tends to resist changes in pH when small to moderate amounts of a
strong acid or strong base is added. For example, a solution that contains 0.50 mol
acetic acid (CH3COOH), and 0.50 mol sodium acetate (NaCH 3COO), in 1.0 L of the
solution is a buffer with a pH of 4.74. When 0.010 mol of a strong acid is added to it,
the pH changes from 4.74 to 4.72, only 0.02 pH unit; adding 0.010 mol of strong base
to this buffer changes the pH from 4.74 to 4.76. These are only slight pH changes,
clearly much less than those that occur when the same number of moles of HCl or
NaOH are in the water, as described above.

Buffer Action
A buffer is a chemical system that maintains a relatively constant pH because
the buffer contains a weak acid that can react with added base, and the buffer also
contains a weak base that can react with added acid. In addition, the acid and base
components of a buffer solution must not react with each other. A conjugate acid-
base pair, such as acetic acid and acetate ion (from sodium acetate), satisfies this
requirement. In a conjugate pair, if the acid and base react with each other they just
produce conjugate base and conjugate acid-no observable change occurs. For
example, acetic acid reacts with acetate ion to form acetate ion and acetic acid.
CH3COOH(aq) + CH3COO-(aq) ⇄ CH3COO- (aq) + CH3COOH (aq)
CONJ ACID CONJ BASE CONJ BASE CONJ ACID

Figure 2.15 pH changes. The pH of water changes dramatically when 0.010 mol of acid (HCl) or
base (NaOH) is added to it. By contrast, the pH of a sodium acetate-acetic acid buffer containing
0.50 mol acetic acid and 0.50 mol sodium acetate remains relatively constant when 0.010 mol HCl or
NaOH is added to it. Only slight change in pH occurs.

A buffer usually consists of approximately equal quantities of a weak acid and

its conjugate base, or a weak base and its conjugate acid.

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The blood of a mammal is an aqueous solution that maintains a constant pH,

that is, a buffer. Consider human blood, whose normal pH is 7.40±0.05. If the pH
decreases below 7.35, a condition known as acidosis occurs; increasing the pH
above 7.45 causes alkalosis. Both conditions can be life-threatening. Acidosis, for
example, causes a decrease in oxygen transport by hemoglobin and depresses the
central nervous system, leading in extreme cases to coma and death by creating
weak and irregular cardiac contractions-symptoms of heart failure. To prevent such
problems, your body must keep the pH of your blood nearly constant.

Carbon dioxide provides the most important blood buffer (but not the only one). In
solution, CO2 reacts with water to form H2CO3, which ionizes to produce H3O+ and
HCO3- ions. The equilibria are

CO2(aq) + H2O(l) ⇄ H2CO3(aq)

H2CO3(aq) + H2O(l) ⇄ H3O (aq) + HCO3-(aq)
+

Since H2CO3 is a weak acid and HCO3- is its conjugate weak base, together they
constitute a buffer. The normal concentrations of H 2CO3 and HCO3- in the blood are
0.0025 M and 0.025 M, respectively – a 1:10 ratio. As long as the ratio of H 2CO3 to
HCO3- concentration remains about 1 to 10, the pH of the blood remains near 7.4.

Example Problem:
Calculate the pH of a buffer containing 0.050 M pyruvic acid, CH 3COCOOH, and
0.060 M sodium pyruvate, NaCH3COCOO. Ka of pyruvic acid = 3.2 x10-3.
Solution: Applying the Henderson-Hasselbalch equation:

pH = pKa + log ¿ ¿ = log (3.2 x10-3) + log ([CH3COCOO-]/[CH3COCOOH])

pH = 2.49 + log (0.060/0.050) =2.49 + log (1.2) = 2.49 + 0.079 = 2.57

Reasonable Answer Check. The concentration of conjugate base is a bit higher than the
concentration of conjugate acid. Therefore, the pH should be slightly higher than the pKa.
Which it is.

References:

1. Campbell, M.K., Farrell, S.O., 2010. Biochemistry, 7th Edition, Belmont, California, USA:
Brooks/Cole

2. Voet,J., Voet, J.D., Pratt, C.W. 2013. Fundamentals of Biochemistry-Life at the Molecular
Level, 4th Edition. Rosewood Drive, Danvers, Ma, USA: John Wiley & Sons Inc.

http://youtu.be/ujkuW-0cpNw
https://youtu.be/LS67vS10O5y

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