Chapter 2: Water, Weak Interactions and Buffers

Objectives:

• Dissect the structure-function relationship of water molecules.

• Characterize the importance of water to biological systems.
• Investigate the ability of water to act as a solvent.
• Explore how the structure of biomolecules is influenced by water.
• Characterize the non-covalent interactions within biomolecules.
• Determine the mechanisms, and importance, of buffers and pH.

Text Readings:
Stryer 2nd or 3rd Editions: All of Chapter 2
 Water
-General-
• Water is the most abundant molecule in living organisms.
• Water has both passive and active roles in biochemistry.

Passive: The structure (hence function) of biomolecules

form in response to interaction with water. For example,
protein folding is driven to bury hydrophobic residues.

Active: Water is a participant in many biochemical

reactions. For example, peptide bond formation releases
a water molecule.
 Water
-Matrix of Life-
• Water is so critical to our understanding of the molecular basis of life
that it shapes the way we look for life.
• While the presence of water on another planet does not ensure life, it is
difficult to imagine life (at least as we know it) in the absence of water.
• Further, the presence of water on other planets is a critical determinant
of their habitability by humans.
• More recently, scientists have started to contemplate alternate liquids,
such as ammonia or formamide, that might also be suitable for life.

formamide
 Water
-Structure/Function Relationship-
• The simple structure of water offers a perfect starting point to illustrate
the structure-function perspective.
• Oxygen and hydrogen differ in their electronegativities.
• Oxygen is more electronegative than hydrogen, giving water a
permanent dipole.
• Oxygen has a partial negative charge and each hydrogen has partial
positive charge.

• The dipole of a water molecule influences it’s ability to:

• form electrostatic interactions with charged molecules.
• form hydrogen bonds (including with other water molecules).
 Hydrogen Bonds
-General-

• Hydrogen bonds are electrostatic

interactions between an electronegative
atom with a hydrogen covalently linked
(donor) to another electronegative atom
with a free electron pair (acceptor).
• Oxygen and nitrogen are common
hydrogen bonders within biomolecules.
• Oxygen and nitrogen can each serve as
hydrogen bond donors and acceptors.

The significance of the hydrogen bond for

physiology is greater than that of any other
single structural feature.
-Linus Pauling
 Hydrogen Bonds
-Strength and Geometry-
• Hydrogen bonds are relatively weak, ~5%
the strength of a covalent bond.
• Hydrogen bonds are about double the length
of a covalent bond.
• The strength of a hydrogen bond depends on
its geometry.
• For example, anti-parallel beta sheets are
more stable than parallel because there is
better geometry of hydrogen bonding.

Parallel Anti-Parallel
 Water
-Unusual Properties-
• Each water molecule can donate and accept two hydrogen bonds.
• Within water, each water molecule has the potential to participate in
four hydrogen bonds with four other water molecules.
• In liquid water, each molecule participates in an average of 3.4
hydrogen bonds in dynamic “flickering clusters”.
• The hydrogen bonds between water molecules confer great internal
cohesion which influences the properties of water.
 Water
-Unusual Properties-

The large number of hydrogen bonds within water contributes to the

high heat of vaporization and specific heat capacity of water.

• Heat of Vaporization: the amount of heat requires to vaporize

a liquid at its boiling temp.

• Specific Heat Capacity: the amount of heat required to raise

the temperature of a substance one degree.
 Water
-Unusual Properties-

• Water has a higher melting point, boiling point, and heat of vaporization
than most common solvents.
 Water
-Unusual Properties-

• Living organisms burn tremendous amounts of energy, a by-product of

which is heat.
• Most living organisms are isothermic, they need to regulate and
maintain their temperatures.
• The high composition of water within our bodies, coupled with the
high specific heat capacity of water, helps us to:
 Water
-Unusual Properties-

• In ice, each water molecule participates in four hydrogen bonds with

other water molecules.
• This ordered arrangement of ice has a lower density than liquid water,
as a consequence, ice floats on water.
 Polywater
-A New, Deadlier Form of H20-
• A Soviet physicist was studying the
Polywater
properties of water forced through quartz
Freezing Temp -40 oC
tubes.
Boiling Temp 150 oC
• This treatment resulted in a new form of Density 1.4 g/cm3
water (polywater) with a higher boiling
Viscosity 15X greater than
point, lower freezing point, and much regular water
higher viscosity than ordinary water.
• Polywater was proposed to result from a novel arrangement of interaction
between water molecules.
 Polywater
-A New, Deadlier Form of H20-
• There was considerable concern that the unusual networking of water
molecules within polywater was self-propagating and could be used as a
weapon.
• "I regard the polywater as the most dangerous material on earth. Even as I
write there are undoubtedly scores of groups preparing polywater. Treat it
as the most deadly virus until its safety is established.”
• It turned out to be B.S. (Bad Science).
• An American scientist demonstrated that his own sweat had properties
remarkable similar to polywater, suggesting the unique properties reflected
the influence of impurities.
 Waters Ability to Act as a Solvent
-Electrostatic Interactions-
• Water molecules can interact, and dissolve, charged solutes through formation
of layers of hydration.
• By virtue of their small size and permanent dipole, water molecules have great
versatility in interacting with both positively and negatively charged ions.
 Waters Ability to Act as a Solvent
-Hydrogen Bonds-
• Biomolecules have functional groups that can form hydrogen bonds.
• These groups can hydrogen bond within the same molecule, other
biomolecules, or with water.
• By virtue of their small size and ability to serve as either donors or
acceptors, water molecules are ideal hydrogen bonding partners.
 Water
-Solubility of Dissolved Molecules-
• The solubility of molecules in water depends on the ability to interact
with water molecules.
• Molecules that carry charge (+ or -) and/or participate in hydrogen
bonds (donors or acceptors) have the greatest solubility in water.
• Hydrophilic (water-loving) molecules are polar.
• Hydrophobic (water-fearing) molecules are non-polar.
• Amphipathic molecules contain both hydrophobic and hydrophilic
portions (e.g. fatty acids).
 Water
-Solubility of Dissolved Molecules-
• Many biologically important gases, such as CO2 and O2, are non-polar
and therefore have limited solubility in water (and blood).
• Their limited solubility presents a challenge for their transport.
• Specialized transport proteins and strategies are required for transport
of CO2 and O2 (to be discussed in a later chapter).
 Water
-Behavior of Amphipathic Substances-
• When an amphipathic molecule is mixed with
water the hydrophilic regions interact
favorably with water but the hydrophobic
regions cluster together to present the smallest
surface to water.
• The forces that hold the non-polar regions of
the molecule together are called hydrophobic
interactions.
• Most biomolecules are amphipathic.
• Hydrophobic drive is a primary driving force
in formation and stabilization of biomolecular
structures.
 Weak Interactions
-Crucial to Molecular Structure and Function-
• Most biomolecules represent stable polymers of covalently linked
building blocks.
• The three-dimensional structures formed by these polymers are
largely determined through non-covalent interactions.
• Interactions between biomolecules are also largely determined by
non-covalent interactions.
• Non-covalent interactions enable:
• Transient, dynamic interactions.
• Flexibility of structure and function.
 Weak Interactions
-Crucial to Molecular Structure and Function-

• Non-covalent forces influence:

• formation and stabilization of structures of biomolecules.
• recognition/interactions between biomolecules.
• binding of reactants to enzymes.

• Non-covalent interactions within biomolecules include:

• hydrogen bonds.
• ionic (electrostatic) interactions.
• hydrophobic interactions.
• van der Waals interactions.
 Weak Interaction
-Hydrogen Bonds-

• Many of the functional groups with biomolecules

have hydrogen bonding capacity.
• These groups can form hydrogen bonds with:
• Water molecules
• Groups in the same molecule (intramolecular)
• Groups in other molecules (intermolecular)
 Weak Interactions
-Hydrogen Bonds-

• Hydrogen bonds are critical for the specificity of biomolecular

interactions but not for the formation of biomolecular structures.
• In the unfolded state, these groups can hydrogen bond with water, a
nearly perfect hydrogen bonder.
• Little is to be gained, from a hydrogen bonding perspective, with
formation of higher order structures.
 Weak Interactions
-Ionic (Electrostatic) Interactions-
• Electrostatic interactions between charged groups
can be attractive (oppositely charged groups) or
repulsive (similarly charged groups).
• The magnitude of contribution of ionic
interactions to biomolecular structures is reduced
by the shielding of these groups by water
molecules.
• Water tends to shield the charged groups, greatly
diminishing the strength of the interaction.
• The strength of electrostatic interactions depends
on the distance separating the atoms and the nature
of the intervening medium.
 Weak Interactions
-van der Waals Forces-
• Interaction between permanent and induced dipoles; short range, low
magnitude interactions.
• When two atoms are separated by the sum of the van der Waals radii,
the attraction is maximal.
• When two surfaces of complementary shapes come together a large
number of atoms are brought into van der Waals contact.
• Abundant in the core of folded proteins.
 Weak Interactions
-Hydrophobic Effect-

• Drive to have polar groups interacting with

water and non-polar regions shielded away from
water.
• For example, in protein folding:
– Non-polar side chains cluster in the interior
of the protein, away from water.
– Polar and charged side chains remain on the
outer surface facing water.
• Notably the folding of a protein involves
creation of a more ordered state, which seems to
be in contradiction of the Second Law
Thermodynamics.
 Weak Interactions
-Thermodynamics of the Hydrophobic Effect-

• The water molecules around hydrophobic molecules are more ordered than they
would be in pure water, as such, the introduction of the non-polar molecule
causes a decrease in the entropy of water.
• The association of non-polar molecules (or regions) releases some of the
ordered water molecules, resulting in an increase in the entropy of water.
• The folding of a polypeptide decreases the entropy of the polypeptide but
increases the entropy of the associated water.
 Does Water have a Memory?

Andy Dufresne: You know what they say about the Pacific?

Red: No.

Andy Dufresne: They say it has no memory.

-The Shawshank Redemption

 Does Water have a Memory?
• A paper reported that an extreme dilution of a biomolecule retained
biological activity.
• The extent of the dilution was such that there was no possibility that even
a single molecule remained.
• The authors suggested that water molecules “remember” what the original
molecule looked like through retention of the interactions that existed
between water molecules in the presence of active agent.
• This was published in Nature, the most respected scientific journal.
• In the following issue, following much uproar, Nature offered: "We
conclude that there is no substantial basis for the claim that (the molecule)
at high dilution retains its biological effectiveness, and that the hypothesis
that water can be imprinted with the memory of past solutes is as
unnecessary as it is fanciful.”
 Bad Science Can be Big Business

• Homeopathic remedies are prepared by repeatedly diluting a chosen

substance; often 30 sequential dilutions of 1 in 100.

• This corresponds to a dilution of 1 in 1, 000, 000, 000, 000, 000, 000,

000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000, 000.

• By our current understanding of the natural world, homeopathy makes no

sense.

• Countless investigations have failed to find any scientific merit to

homeopathy and yet selling water as medicine remains a multi-billion
dollar industry.

• The disproven theory that water molecules remember the shapes of

molecules has been used as support of homeopathy.
 Ionization of Water

• In solution, the structure of water is more complicated than H2O.

• Water has a limited tendency to ionize to hydrogen ions (H+) and
hydroxide ions (OH-).
H2O ßà H+ + OH-

Keq = [H+][OH -] / [H2O] = 1.8 x 10-16 M

and [H2O] = 55.5 M

therefore 1.8 x 10-16 M x [55.5 M] = [H+][OH-]

Kw = [H+][OH-] =1.0 X 10-14 M2

Kw is the ion product of water.

 The pH scale
It is more convenient to express [H+] as pH.

pH = -log [H+] = log 1/[H+]

e.g. [H+] = 1 X 10-5 M or 0.00001 M

pH = -log [10-5]
pH = 5

e.g. [H+] = 1 X 10-6 M or 0.000001 M

pH = -log [10-6]
pH = 6

• pH is a log scale such that the difference of 1 pH

unit equals a 10-fold difference in [H+].
 Weak Acids and Bases have Characteristic Dissociation
Constants
• Strong acids and bases dissociate completely in water.
• Weak acids and bases do not dissociate completely in H2O and the extent of
dissociation can be quantified.

Ka = [H+][CH3COO-] / [CH3COOH]

• Ka values often expressed as pKa’s (pKa = -log Ka).

Titration Curves Reveal the pKa of Weak Acids
• The ratio of the acid to the conjugate
base changes over the course of the
titration curve.

• When pH = pKa then [A-] = [HA].

• When pH = pKa the solution is best able

to resist changes in pH.

• Buffering region extends one pH unit on

either side of the pKa point.
• For a weak acid of pKa 4.76, the
buffering range would be 3.76 to
5.76.
Titration Curves Reveal the pKa of Weak Acids

• The line in red represents the weakest acid.

• The line in blue represents the strongest acid.

• The lower the pKa, the stronger the acid.

Molecules can have Multiple Ionizing Groups
 Buffers are Important to Biological Systems

• Organisms need to be able to maintain a constant pH.

• Changes to pH could alter the protonation state of biomolecules,
potentially changing their structure and function.
• A number of weak acids that serve to buffer biological systems.
For example, the pH within blood is maintained by a
bicarbonate buffer system.

• Compensatory respiratory alkalosis serves to maintain the ratio

of H2CO3/HCO3- to maintain a constant pH.
 The Henderson-Hasselbalch Equation
• Describes the relationship between:
(1) the pH of the solution.
(2) the pKa of the weak acid
(3) the relative concentrations of the weak acid (HA) and conjugate base (A-)

• Given any two of these variables it is possible to calculate the third.

 Henderson-Hasselbalch Sample Questions

1) Calculate the pH of a mixture of 0.01 M acetic acid and 0.1 M sodium

acetate. pKa of acetic acid is 4.76

pH = pKa + log [A-] / [HA]

pH = 4.76 + log 0.1 / 0.01
pH = 4.76 + 1.0 = 5.76

2) Calculate the pKa of lactic acid given that a mixture of 0.01 M lactic acid and
0.087 M lactate has a pH of 4.80.

pH = pKa + log [A-] / [HA]

pKa = pH – log [0.087] / [0.01]
pKa = 4.80 – 0.94 = 3.86
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