Biochemistry by Campbell and Farrell (8E)

Chapter Two: Water: The Solvent for Biochemical Reactions

2.1. Water and Polarity

Water is the principal component of cells. The geometry of the water molecule (bent geometry)
and its properties as a solvent play major roles in determining the properties of living systems.
Electronegativity is the tendency of an atom to attract electrons to itself in a chemical bond in other
words, to become negative.
2.1.1. What is polarity?
Polarity is the phenomenon by which one atom is rich in electron while the other is lacking
electrons brought by the difference in electronegativity of the atoms involved in the chemical bond.
In situations wherein the electronegativity difference is quite small, the sharing of electrons in the
bond is very nearly equal, and the bond is essentially nonpolar.
The bonds in a molecule may be polar, but the molecule itself can still be nonpolar because of its
geometry.
Bonds with positive and negative ends are called dipoles.
2.1.2. Why do some chemicals dissolve on water while others dont?
The polar nature of water largely determines its solvent properties.
Ionic compounds with full charges, such as potassium chloride (KCl; K+ and Cl in solution), and
polar compounds with partial charges (i.e., dipoles), such as ethyl alcohol (C2H5OH) or acetone
[(CH3)2C=O], tend to dissolve in water, while less polar molecules tend not to dissolve as readily
in water, if at all.
The underlying physical principle is electrostatic attraction between unlike charges, but there are
different types of bonds with different strengths depending on these electrostatic attractions.
Ionic bonds and covalent bonds are the strongest bonds, being many times stronger than the next
weakest one.
Similar to ionic bonds found in salt crystals, biomolecules often have ionizable groups on them.
o Salt bridge - an interaction that depends on the attraction of unlike charges
Ions in solution can also interact with molecules that have dipoles.
 There are several types of weak forces that are called van der Waals forces, after the Dutch scientist
Johannes Diderik van der Waals. Included in these forces are three noncovalent bonds that do not
involve an electrostatic interaction of a fully charged ion.
Dipoledipole interactions. These forces occur between molecules that are dipoles, with the partial
positive side of one molecule attracting the partial negative side of another molecule.
Dipole-Induced Dipole Interactions. A permanent dipole in a molecule when it comes into close
contact with any molecule, even those that have no dipoles, can induce a transient dipole in the
other. As the electron cloud of the dipole pushes against the electron cloud of the other molecule,
it momentarily distorts the electron cloud. This creates a brief dipole and, in that moment, the two
molecules are attracted to one another.
Induced dipoleinduced dipole interactions. In the same way that a dipole can create a momentary
dipole in another molecule, any two molecules can effectively do the same thing. When two
molecules lacking dipoles bump into each other, they distort each others electron cloud, thereby
creating a brief interaction between these induced dipoles. This attractive force is often referred to
as a London dispersion force. This type of force is the reason that all molecules are attracted to
one another to a very small degree, and explains why nonpolar molecules would have attractions
for one another.
o London dispersion force - an attraction between transient induced dipoles.
Water is a very polar molecule, and it easily makes iondipole bonds with ionic compounds, or
dipoledipole bonds with polar neutral compounds.
If the bonds formed between the dissolving ions and water are more favorable than the bonds
holding the solid together, it will dissolve.
Ionic and polar substances tend to dissolve in water, and they are referred to as hydrophilic (water-
loving, from the Greek) for this tendency.
Nonpolar molecules do not dissolve in water and are referred to as hydrophobic (water-hating,
from the Greek).
A nonpolar liquid forms a two-layer system with water; an example is an oil slick. The interactions
between nonpolar molecules are called hydrophobic interactions or, in some cases, hydrophobic
bonds.
2.1.3. Why do oil and water, mixed together separate into layers?
 A single molecule may have both polar (hydrophilic) and nonpolar (hydrophobic) portions.
Substances of this type are called amphipathic.
A long-chain fatty acid having a polar carboxylic acid group and a long nonpolar hydrocarbon
portion is a prime example of an amphipathic substance. The carboxylic acid group, the head
group, contains two oxygen atoms in addition to carbon and hydrogen; it is very polar and can
form a carboxylate anion at neutral pH. The rest of the molecule, the tail, contains only carbon
and hydrogen and is thus nonpolar. A compound such as this in the presence of water tends to form
structures called micelles, in which the polar head groups are in contact with the aqueous
environment and the nonpolar tails are sequestered from the water.
2.2. Hydrogen bonds
Hydrogen bonding is of electrostatic origin and can be considered a special case of dipole-dipole
interaction.
Hydrogen bonds are much weaker than normal covalent bonds.
When hydrogen is covalently bonded to a very electronegative atom such as oxygen or nitrogen,
it has a partial positive charge due to the polar bond, a situation that does not occur when hydrogen
is covalently bonded to carbon.
This partial positive charge on hydrogen can interact with an unshared (nonbonding) pair of
electrons (a source of negative charge) on another electronegative atom. All three atoms lie in a
straight line, forming a hydrogen bond. This arrangement allows for the greatest possible partial
positive charge on the hydrogen and, consequently, for the strongest possible interaction with the
unshared pair of electrons on the second electronegative atom.
The group comprising the electronegative atom that is covalently bonded to hydrogen is called the
hydrogen-bond donor, and the electronegative atom that contributes the unshared pair of electrons
to the interaction is the hydrogen-bond acceptor.
Hydrogen bonds have a vital involvement in stabilizing the three-dimensional structures of
biologically important molecules, including DNA, RNA, and proteins. The hydrogen bonds
between complementary bases are one of the most striking characteristics of the double-helical
structure of DNA.
Transfer RNA also has a complex three-dimensional structure characterized by hydrogen-bonded
regions. Hydrogen bonding in proteins gives rise to two important structures, the -helix and -
pleated sheet conformations. Both types of conformation are widely encountered in proteins.
2.3. Acids, Bases, and pH
The biochemical behavior of many important compounds depends on their acidbase properties.
2.3.1. What are acids and bases?
An acid is a molecule that acts as a proton (hydrogen ion) donor. (Bronsted Acid)
A base is a molecule that acts as a proton (hydrogen ion) acceptor. (Bronsted Base)
How readily acids or bases lose or gain protons depends on the chemical nature of the compounds
under consideration.
Acid Strength is a numerical expression of the amount of hydrogen ion released when a given
amount of acid is dissolved in water.
o Acid strength is the tendency of an acid to dissociate to a hydrogen ion and its conjugate
base.
Acid dissociation constant (Ka) is a number that characterizes the strength of an acid which can be
written for any acid, HA, that reacts according to the equation

HA H+ + A-
ACID CONJUGATE BASE

[+ ][ ]
=
[]

2.3.2. What is pH?

The tendency of water to dissociate to give hydrogen ion and hydroxide ion is measured as the
ion product constant for water.
o Kw, the ion product constant for water, is equal to 10-14 at 25C.
o Kw = [H+] [OH-]
pH (the potential of hydrogen or sometimes called as the power of hydrogen) is a quantity
expressing the wide range of possible hydrogen ion and hydroxide ion concentrations in aqueous
solution.
o pH = -log [H+]

PRACTICE

Calculate the pH of the following aqueous solutions:

 1. 1 x 10-3 M HCl Ans: pH = 3.0
2. 1 x 10-4 M NaOH Ans: pH = 10.0
3. 0.100 M Ba(OH)2 Ans: pH = 13.301
When a solution has a pH of 7, it is said to be neutral, like pure water. Acidic solutions have pH
values lower than 7, and basic solutions have pH values higher than 7.
In biochemistry, most of the acids encountered are weak acids. These have a Ka well below 1. To
avoid having to use numbers with large, negative exponents, a similar quantity, pKa, has been
defined by analogy with the definition of pH:

pKa = -log Ka

The pKa is a more convenient numerical measure of acid strength. The smaller its value, the
stronger the acid. This is the reverse of the situation with Ka, where larger values imply stronger
acids.
2.3.3. Why do we need to know pH?
An equation connects the Ka of any weak acid with the pH of a solution containing both that acid
and its conjugate base. This relationship has wide use in biochemistry, especially where it is
necessary to control pH for optimum reaction conditions.
Some reactions cannot take place if the pH varies from the optimum value. Important biological
macromolecules lose activity at extremes of pH.
HENDERSON-HASSELBALCH EQUATION

[ ]
pH = pKa + log
[]

When a solution contains equal concentrations of a weak acid and its conjugate base, the pH of
that solution equals the pKa value of the weak acid.
2.4. Titration curves
Titration is an experiment in which measured amounts of base are added to a measured amount of
acid.
The point in the titration at which the acid is exactly neutralized is called the equivalence point.
Near the inflection point, the pH changes very little as more base is added.
When the pH of a solution is less than the pKa of an acid, the protonated form predominates.
 When the pH of a solution is greater than the pKa of an acid, the deprotonated (conjugate base)
form predominates. pH < pKa.

PRACTICE

Calculate the pH at the following points when 1 mol of acetic acid is titrated with sodium
hydroxide. (Ka acetic acid = 1.76 x 105)

1. 0.1 mol NaOH added Ans: pH = 3.81

2. 0.3 mol NaOH added Ans: pH = 4.39
3. 0.5 mol NaOH added Ans: pH = 4.76
4. 0.7 mol NaOH added Ans: pH = 5.13
5. 0.9 mol NaOH added Ans: pH = 5.71
2.5. Buffers
Buffer solutions tend to resist changes in pH when small to moderate amounts of a strong acid or
strong base are added.
A buffer solution consists of a mixture of a weak acid and a conjugate base.
2.5.1. How do buffers work?
Buffers work because they obey Le Chateliers principle. This principle states that if stress is
applied to a system in equilibrium, the equilibrium will shift in the direction that relieves the stress.
Therefore, if we add hydrogen ion to a buffer system, we have added stress to the reaction:

HA H+ + A-

To relieve the stress, the H+ reacts with the A to maintain the equilibrium.
Effect of adding an acid in a buffer solution showed in Henderson-Hasselbalch equation

[ ][ + ]
pH = pKa + []+[ +
]

PRACTICE

Calculate the pH if you added 3 mL of 0.1 M HCl to (a) 97 mL pure water at pH 7.0 or
(b) 100.0 mL of a H2PO4-/HPO4-2 buffer solution at pH 7.0. (Ka H2PO4- = 6.31 x 10-8)
 Ans: a) pH= 2.52; b) pH = 6.97

2.5.2. How do we choose a buffer?

The rule of thumb is that the pKa should be 1
pH unit from the pH of the reaction; pH unit
is even better.
Although the perfect generic buffer would have a
pH equal to its pKa, if the reaction is known to
produce an acidic product, it is advantageous if
the pKa is below the reaction pH, because then the
buffer capacity increases as the reaction
proceeds.
The following criteria are typical for selecting a buffer for a biochemical reaction:
1. Suitable pKa for the buffer.
2. No interference with the reaction or with the assay.
3. Suitable ionic strength of the buffer.
4. No precipitation of reactants or products due to presence of the buffer.
5. Non-biological nature of the buffer.
Buffering capacity is the measure of the amount of acid or base that can be absorbed by a given
buffer solution.
o A buffer solution with low concentrations of both the acid and base forms is said to
have a low buffering capacity.
o A buffer that contains greater amounts of both acid and base has a higher buffering
capacity.
If a buffer solution contained a suitable ratio of acid to base, but very low concentrations of both,
it would take very little added acid to use up all of the base form, and vice versa.
2.5.3. How do we make buffers in the laboratory?
To have a buffer, all that is necessary are the two forms of the buffer present in the solution at
reasonable quantities.
o This situation can be obtained by adding predetermined amounts of the conjugate base
form (A) to the acid form (HA), or we could start with one and create the other,
which is how it is usually done in practice.
 Depending on the relationship of the pH we desire to the pKa of the buffer, it may be more
convenient to start with one than the other.
For example, if we are making an acetic acid/acetate buffer at pH 5.7, it would make more sense
to start with the A form and to add a small amount of HCl to bring the pH down to 5.7, rather
than to start with HA and to add much more NaOH to bring the pH up past the pKa.
2.5.4. Are naturally occurring buffers present in living organisms?
The H2PO4/HPO4 pair is the principal buffer in cells.
o This is because physiological pH in most organisms stays at pH 7 and this phosphate
buffer system is suitable in near pH 7.2.
In blood, phosphate ion levels are inadequate for buffering, and a different system operates.
The buffering system in blood is based on the dissociation of carbonic acid (H2CO3) where the
pKa of H2CO3 is 6.37. The pH of human blood, 7.4, is near the end of the buffering range of this
system, but another factor enters into the situation.
Carbon dioxide can dissolve in water and in water-based fluids, such as blood. The dissolved
carbon dioxide forms carbonic acid, which, in turn, reacts to produce bicarbonate ion. The
conversion of aqueous CO2 and water to carbonic acid is catalyzed by the enzyme, carbonic
anhydrase. This is one of the most efficient enzymes in biochemistry, capable of converting a
million molecules of CO2 to carbonic acid per second.
At the pH of blood, which is about one unit higher than the pKa of carbonic acid, most of the
dissolved CO2 is present as HCO3 . The CO2 being transported to the lungs to be expired takes
the form of bicarbonate ion. A direct relationship exists between the pH of the blood and the
pressure of carbon dioxide gas in the lungs. The properties of hemoglobin, the oxygen-carrying
protein in the blood, also enter into the situation.
The phosphate buffer system is common in the laboratory (in vitro, outside the living body) as
well as in living organisms (in vivo).
The buffer system based on TRIS [tris(hydroxymethyl)aminomethane] is also widely used in
vitro. Other buffers that have come into wide use more recently are zwitterions, which are
compounds that have both a positive charge and a negative charge. Zwitterions are usually
considered less likely to interfere with biochemical reactions than some of the earlier buffers.
Alkalosis is a condition in which blood pH rises above 7.45.
Acidosis is a condition in which blood pH drops below 7.35