PRINCIPLES OF

BIOCHEMISTRY
• BIOC -3(3-0)
• Learning Aim
• Understanding of chemical basis of working of
• bio molecules
• Recommended Books
• (1) Essentials Of Medical Biochemistry
• By Mushtaq Ahmad Volume 1
• (2) Lehninger Principles of Biochemistry
• By D L Nelson and M M Cox.
Ionic Product Of Water And Its
relationship With pH

2
 Hydrogen Ion Concentration &
pH
• Water is most abundant substance in the
human body making up to 60% to 70% of
the body mass.
• Water is dipolar molecule, Hydrogen bonds
exists between water molecules making it a
liquid at room temperature and give solvent
properties. Pure water is very slightly
dissociated (weak electrolyte).At25 C 1/10
million molecules in pure water is ionized. 3
 Ionic Product of Water & pH

H2O H+ + OH-

[H+] [OH-]
Keq = = 1.8x10-16 M
[H2O]

Concentration of “water in water” ([H2O]) is 55.6 M [next slide], thus

Kw = [H+] [OH-] = 10-14 M2 Constant ion product!

Pure water has equal quantities of H+ and OH- ions, or, put differently,
pure water has equal [H+] and [OH-].

[H+] = [OH-] = 10-7 M = 0.1 mM

 Molarity Of Water

Concentration is measured in moles per liter (mol/l) or simply M.

1 l = 1,000 ml of water has a mass of 1,000 gr.

1 mole of water has a mass of 18 gr (hydrogen 1 Da, oxygen 16 Da).

Thus 1 liter of water (1,000 gr) contains 1,000 gr / 18 gr moles of water.

[H2O] = (1,000 gr / 18 gr) M = 55.6 M.

 +
SOURCE: http://en.wikipedia.org/wiki/Image:PH_scale.png#file
[H ] M

10
0 A strong acid pH
-1
10
-2
• Most living cells have a very narrow
10 +
-3 range of tolerance for pH, i.e. [H ].
10

10
-4 • [H+] is controlled in all biological
10
-5 organisms, and in virtually all biochemical
-6
experiments.
10
10
-7 • Each pH unit represents
+ a factor of 10
difference in [H ]
• The pH effects the structure & activity of
-8
10

-9
10 the biological macromolecules;for example
10
-10 the catalytic activity of enzymes is strongly
10
-11 depentent on pH.
-12
10
-13
A strong base
10
-14
10

+ - -14 7
The pH scale goes from 0 to 14—because [H ][OH ] = 10
pH profiles of enzymatic reactions
Amylase

Pepsin

UCI Bio199 Independent Research

 Base: proton acceptor
The pH scale

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

neutral/pure water has

[H+] = [OH-] = 10-7 M, so

pH = -log(10-7) = -(-7) = 7

Acid: proton donor

 Strong acids and bases
pH = -log [H+]

HCl H+ + Cl-

HCl is a strong acid that completely dissociates in

water. 1 M HCl will thus yield 1 M [H+] and the pH will be
pH = -log [H+] = -log(1) = 0

NaOH is a strong base that completely dissociates in

water. 1 M NaOH will thus yield 1 M [OH-]. Since
[H+] [OH-] = 10-14 M and must remain constant
[H+] = 10-14 M and the pH will be
pH = -log [H+] = -log(10-14) = 14

Life is compatible only in a narrow pH range around pH 7.

 The Conceptual Problem with pH
• Because it’s a logarithmic scale, it doesn’t make
“sense” to our brains.
• Every factor of 10 difference in [H+] represents 1.0
pH units, and
• Every factor of 2 difference in [H+] represents 0.3
pH units.
• Therefore, even numerically small differences in
pH, can have profound biological effects…

11
 How Can You Actually
Determine the pH of a Solution?
• Use a pH meter—read the number.
• Use pH paper (color patterns indicate pH).
• Titrate the solution with precise amounts of
base or acid in conjunction with a soluble dye,
like phenolphthalein, whose color changes
when a specific pH is reached.

8
BUFFER SOLUTION
An aqueous solution system which
tends to maintain the pH of the
solution when small amount of acid
or base is added to it .
 Buffers
Buffers
• resist changes in pH from the addition of
acid or
base
• in the body absorb H3O+ or OH from
foods and cellular processes to maintain
pH
• are important in the proper functioning of
cells and blood 14
 Buffers (continued)
When an acid or
base
is added
• to water, the pH
changes
drastically
• to a buffer
solution, the pH
does not change
very much; pH is
15
 BIOLOGICAL BUFFER
SYSTEMS
• Bicarbonate buffer (53%)
• Hemoglobin (35%)
• Plasma proteins (7%)
• Phosphate (5%)
 How does a buffer work?

At the pKa, [HAc] = [Ac-] so the system is able to absorb the addition of HO-
or H+. If we add HO- near the pH where [Hac] = [Ac-] (ie pH ~= pKa) then
HAc can release H+ to offset the HO- added but the ratio of HAc to Ac- does
not change much. If we add H+ then Ac- can absorb H+ to form HAc.
Hence, the pH does not change much.
Titration curves
[H+] [Ac-]
Ka =
[HAc]

[Ac-]
pH = pKa + log
[HAc]

2 Start at low pH and begin to add HO-.

The product of [H+] [HO-] must
remain constant, so adding HO-
means [H+] must decrease and thus
pH increases. At the pKa, [Ac-] and
[HAc] are equal, so adding more HO-
does not change the ratio of [Ac-] to
[HAc] very much and thus the pH
does not change very much (shallow
slope of titration curve from ~1 pH
unit below pKa to ~1 pH unit above).
Buffers are vitally important in biochemical systems
since pH needs to be controlled. Living systems must
be “buffered” to resist large variations in pH.

Phosphate
H3PO4 H+ + H2PO4- pKa1 = 2.2
H2PO4- H+ + HPO42- pKa2 = 7.2
HPO42- H+ + PO43- pKa3 = 12.7

Carbonate
CO2 + H2O H2CO3
H2CO3 H+ + HCO3- pKa1 = 6.4
HCO3- H+ + CO32- pKa2 = 10.2
Phosphate buffering
Measuring pKa values

NH4+ H+ + NH3

[H+] [NH3]
Ka =
[NH4+ ]

pKa = pH when [NH4+] = [NH3]

Carbon dioxide - carbonic acid - bicarbonate buffer

If blood pH drops due to metabolic

production of H+ then [H2CO3] increases
by protonation of HCO3-, H2CO3 rapidly
loses water to form CO2(aq), which is
expelled as CO2(g).

If the blood pH rises, [HCO3-] increases

by deprotonation of H2CO3, then
breathing rate changes and CO2(g) is
converted to CO2(aq) and then to H2CO3
in the capillaries in the lungs.

What happens to blood pH when you hyperventilate?

What happens to blood pH when you hypoventilate?
 The Henderson-Hasselbalch Equation
[H+] [Ac-]
HAc H+ + Ac- Ka =
[HAc]

[Ac-]
take the -log on both sides -log Ka = -log [H+] -log
[HAc]

[Ac-]
apply p(x) = -log(x) pKa = pH -log
[HAc]

and finally solve for pH…

[Ac-] [Proton acceptor]

pH = pK + log = pKa + log
a
[HAc] [Proton donor]
 Acetic acid has a pKa of 4.8. How many ml of 0.1 M
acetic acid and 0.1 M sodium acetate are required to
prepare 1 liter of 0.1 M buffer with a pH of 5.8?
Substitute the values for the pKa and pH into the Henderson-Hasselbalch
equation:

5.8 = 4.8 + log [Acetate]

[Acetic acid]
1.0 = log [Acetate] 10x then *[Acetic acid]
[Acetic acid] on both sides

10 [Acetic acid] = [Acetate]

For each volume of acetic acid, 10 volumes of acetate must be added (total
of 11 volumes).

Acetic acid needed: 1/11 x 1,000 ml = 91 ml

Acetate needed: 10/11 x 1,000 ml = 909 ml
 What is the pH of 0.15 M acetic acid?

The pKa of acetate is 4.8, so the Ka = 10-4.8 M = 1.58x10-5 M.

O O [H+] [A-] and

_________
R-C-OH R-C-O- + H+ Ka = [H+]=[A-]
[HA]=0.15-[H+]
[HA]
[H+]2 [H+]2
Ka = ––––– = ––––––––– = 1.58x10-5 M
[HA] 0.15 M - [H+]

[H+]2 +1.58x10-5 M [H+] + (-2.37x10-6 M2) = 0 (ax2+bx+c = 0)

ax2 + b x + c = 0 Quadratic Formula

[H+] = 1.53x10-3 M and thus pH = 2.8

Your 199 prof asks you to make a pH 7 buffer. You already
have 0.1 M KH2PO4. What concentration of K2HPO4 do you
need?

KH2PO4 H2PO4- + K+ and K2HPO4 HPO42- + 2 K+

H2PO4- HPO42- + H+ pKa = 6.86

[HPO42-]
pH = 7 = pKa + log
[H2PO4-]

7 = 6.86 + log(x / 0.1 M)

0.14 = log(x / 0.1 M)
100.14 = x / 0.1 M

x = 0.138 M = [K2HPO4]
 Another HH calculation
Make 200 ml of 0.1 M Na acetate buffer pH 5.1, starting
with 5.0 M acetic acid and 1.0 M NaOH.

Strategy
1. Calculate the total amount of acetic acid needed.
2. Calculate the ratio of the two forms of acetate (A- and
HA) that will exist when the pH is 5.1.
3. Use this ratio to calculate the % of acetate that will be in
the A- form.
4. Assume that each NaOH will convert one HAc to an Ac-.
Use this plus the % A- to calculate the amount of NaOH
needed to convert the correct amount of HAc to Ac-.
(1) How much acetic acid is needed?
200 ml x 0.1 mol/l = 200 ml x 0.1 mmol/ml = 20 mmol
5.0 mol/l x x ml = 5.0 mmol/ml x x ml = 20 mmol
x = 4.0 ml of 5.0 M acetic acid are 20 mmol

(2) What is the ratio of Ac- to HAc at pH 5.1?

[Ac -]
pH = pKa + log HH equation
[HAc]
5.10 - 4.76 = log[A-]/[HAc], thus [Ac-]/[HAc] = 2.19 / 1

(3) What fraction of total acetate is Ac- at pH 5.1?

[Ac-] [Ac-] 2.19

–––– = 2.19; –––––––––– = ––––––– = 0.687 or 68.7%
[HAc] [HAc] + [Ac-] 1 + 2.19
(4) How much OH- is needed to obtain 68.7% Ac-?

Na+ + OH- + HAc  Na+ + Ac- + H2O

mmol NaOH = 0.687 x 20 mmol = 13.7 mmol
1.0 mol/l x x ml = 1.0 mmol/ml x x ml = 13.7 mmol
x = 13.7 ml of 1.0 M NaOH

(5) Final answer (Jeopardy…)

• 4.0 ml of 5.0 M acetic acid

• 13.7 ml of 1.0 M NaOH
• Bring to final volume of 200 ml with water
(ie add about 182.3 ml of H2O).