1

1. INTRODUCTION

Biochemistry is the study of living organisms at the molecular level. One can assume organisms as
complex chemical systems. Organisms contain all the information necessary to grow and to reproduce at
the expence of energy and raw materials from the environment. Biochemistry is a combination of two
major sciences, chemistry and biology.

The chemical make-up of organisms are dictated partly by the (i) availability of raw material e.g
relative abundance of oxygen (O) is 47% oxygen atoms per total # of atoms in earths crust and 25.5 %
oxygen atoms per total # of atoms in human body. Other most abundant elements of earths crust are
silicon, Si (28 %), aluminum, Al (7.9 %), iron, Fe (4.5 %). In contrast the most abundant elements in
living organisms are besides oxygen, hydrogen, H (63 %), carbon, C (9.5 %), nitrogen, N (1.4 %). These
values show that availability of raw material is not the only factor. (ii) fitness of atoms and molecules for
specific roles in life processes. e.g. O, H, C and N are the smallest atoms which form more stable covalent
bonds than any other elements with the same valances. In addition O, N and C are the only elements
that readily form strong multiple bonds.


e.g. C=C CC C=O C=N CN:


Carbon forms a variety of stable molecules with H, O and N. With O2 molecule C forms CO2, it is
a stable gas, soluble in water. This gives rise the circulation of C between organisms. O2 itself is also
soluble in water, consequently it is readily available to nearly all organisms.

1.1. Chemical bonds in living matter

The atoms of living matter are combined with covalent bonding. There are also noncovalent bonds
in the biological systems. These are (a) Ionic bonds result from the electrostatic attraction between two
ionized groups of opposite charge.


e.g. COO
-
---------------------------H3N
+

carboxyl group amino group
of one biomolecule of another biomolecule

(b) Hydrogen bonding results from electrostatic attraction between an electronegative atom (O or N) and
a H atom that is bonded covalently to a second electronegative atom.
H

N-H............O=C OR O-H .........O=C OR O-H..........O-H
| | | | | |
electron electron rich (e.r.) e.d. e.r. e.d. e.r.
defficient(e.d.)

(c) Van der Waals bonds are short-range attractive forces between chemical groups in contact. These
bonds are weaker than ionic and hydrogen bonding. These forces arise from the motion of electrons in
atoms and molecules. They are present in all matter.

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(d) Hydrophobic interactions result from the solvent properties of water.

Ovvvvvvvvvvvvvvvv
| |
hydrophilic head hydrophobic tail

Hydrophobic tail escapes from water and this gives rise the micelle formation in aqueous medium. In
living systems, hydrophobic interactions are primarily responsible for the stability of protein molecules,
membranes and most other subcellular structures.

1.2. Functional groups

The carbon containing species in biochemistry are organic compounds. These are

Alcohol R-OH Aldehyde R-C=O

H

Ketone R-C=O carboxylic acid R-C=O

R OH

thiol(sulfhydryl) R-SH

primary, secondary and tertiary amines RNH2, RNHR, RNRR

Biochemical reactions involve specific chemical bonds and parts of molecules called functional
groups. Specific bonds or linkages:

O
-
O
-

( | | | | |
C-O-C=O C-O-C N-C=O O=P-O-P=O
( ( | | | | |
O
-
O
-


ester ether amide phosphoanhydride
Functional groups:

R O
-

| | |
OH C=O C=O C=O
hydroxyl acyl carbonyl carboxylate





3
O
-
O
-

| |
SH NH2 or NH3
+
O-P=O P=O
| |
O- O
-

thiol amino phosphate phosphoryl


1.3. Water and 30 small-molecule precursors:

Organisms are built from water and thirty small precursor molecules. Water is an excellent solvent
for ionic and polar substances since it is a polar substance. But it is immiscible with nonpolar substances.
Water is a nucleophile (nucleus lover). Nucleophiles seek positively charged or electron defficient
species. Water forms hydrogen bonding with each other and other molecules. Water undergoes self-
ionization.

H2O + H2O H3O
1+
+ OH
1-


Water is the most common compound in living organisms, accounting for at least 70 % of the weight of
most cells. It is the major solvent and also reactant in many biochemical reactions.


Precursors are as follows:

1. 20 L-amino acids: They are building blocks of proteins. Amino acids are joined by peptide bonds to
form a polypeptide chain.
2. 5 aromatic bases: These are two purine bases, adenine (A) and guanine (G) and three pyrimidine bases,
cytosine (C), uracil (U) and thymine (T). They are linked to the sugars, ribose-phosphate or deoxyribose-
phosphate to form nucleotides, the monomeric units of ribonucleic acid (RNA) or dioxyribonucleic acid
(DNA). The nucleotides are joined by phosphodiester bonds to form nucleic acids.
3. 2 sugars: D-glucose is the major product of plant photosynthesis and is a central intermediate in
metabolism. Glucose molecules are joined by glycosidic bonds to form polysaccharides. Another sugar,
D-ribose is the precursor of sugar-phosphate in nucleotides.
4. Palmitate, a fatty acid, glycerol, a tri-alcohol and choline, an amine are the constituents of the
phospholipid molecules that form the matrix of the biological membranes.

1.4. The hierarchy of biological structure

Organisms (animals, plants and bacteria)
|
Organs (liver, skin, lungs, heart etc.)
|
Tissues
|
Cells
|
Organelles (nuclei, mitochondria, chloroplasts, golgi bodies etc)
|
Supramolecular assemblies (noncovalent bonding of macromolecules to form multienzyme complexes,
ribosomes, chromosomes, membranes, structural elements, contractile systems)
Particle mass:10
6
-10
10


4
|
Macromolecules (proteins, nucleic acids, polysaccharides and lipids)
Molecular mass:10
4
-10
9
)
| | | | |
Monomeric units (amino acids, nucleotides, sugars fatty acids+glycerol)
Unit mass:100-350
|
Metabolic intermediates (pyruvate, malate, citrate, glycerol 3-phosphate etc)
Masses:50-100
|
Precursors from environment (CO2, water, ammonia, nitrogen)
Masses:18-44

1.5. Biopolymers

Macromolecules such as proteins, nucleic acids, polysaccharides are biopolymers. Proteins are
polymers of amino acids, nucleic acids are polymers of nucleotides, polysaccharides are polymers of
monosaccharides. Lipids do not exactly fit the definition of polymers, but since lipid molecules are often
found in association with each other, it is sometimes useful to treat lipids as polymers.
O

aaaaaa nucleotide O P O nucleotide
| |
peptide bond (a covalent bond) O-
phosphodiester bond
(covalent bonds)

glucoseOglucose
| |
glucosidic bond (covalent bond)


1.5.1. Proteins

Proteins are polymers of amino acids. There are 20 amino acids that are incorporated into proteins
in all cells. The general structure of amino acids is as follows:


H
|
H3N
+
C
*
COO
-

|
R

Glycine (gly), alanine (ala), valine (val), leucine (leu), isoleucine (ile), proline (pro) are amino
acids with aliphatic R groups. Phenylalanine (phe), tyrosine (tyr), Tryptophan (trp) have aromatic R
groups. Serine (ser), threonine (thr) are alcohols. Histidine (his), lysine (lys), arginine (arg) are bases.
Asparagine (asn), glutamine (gln) are amides. Aspartate (asp), glutamine (glu) are acids. Methionine
(met), cystein (cys) are sulfur-containing amino acids. They are all o-amino acids except proline. Proline
is an o-imino acid. L-amino acids are known to occur in proteins. With the exception of glycine, amino
acids can exist in different stereoisomeric configurations. Amino acids exhibit acid-base properties due to
the presence of o- carboxyl, and o- amino groups, and if any, ionizable R- groups.

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Naturally occurring amino acids, their abbreviations, and structural formulas
* Essential amino acids
Ala = alanine
CH3CH(NH2)COOH
Arg = arginine
H2N-C(=NH)NHCH2CH2CH2CH(NH2)COOH
Asn = asparagine
H2N-C(=O)CH2CH(NH2)COOH
Asp = aspartic acid
HOOC-CH2CH(NH2)COOH
Cys = cysteine
HS-CH2CH(NH2)COOH
Gln = glutamine
H2N-C(=O)CH2CH2CH(NH2)COOH
Glu = glutamic acid
HOOC-CH2CH2CH(NH2)COOH
Gly = glycine
H2N-CH2COOH
His = histidine *

Ile = isoleucine *
CH3CH2CH(CH3)CH(NH2)COOH
Leu = leucine *
CH3CH(CH3)CH2CH(NH2)COOH
Lys = lysine *
H2N-CH2CH2CH2CH2CH(NH2)COOH
Met = methionine *
CH3-S-CH2CH2CH(NH2)COOH
Phe = phenylalanine *

Pro = proline

Ser = serine
HOCH2CH(NH2)COOH
Thr = threonine *
CH3CH(OH)CH(NH2)COOH
Trp = tryptophan *

Tyr = tyrosine

Val = valine *
CH3CH(CH3)CH(NH2)COOH

The pH at which zero net charge exists is called isoelectric pH and is symbolized as pI. The pI is
estimated as follows:
pI=( pK1+pK2)/2 for amino acids with neutral R-groups
pI=(pK1+pKR)/2 for amino acids with acidic R-groups
pI=(pK2+pKR)/2 for amino acids with basic R-groups

pK values of amino acids
pK1 pK2 pKR
Amino acid -COOH -NH3
+
R-group

gly 2.34 9.60
ala 2.34 9.69
val 2.32 9.62
leu 2.36 9.68

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ile 2.36 9.68
ser 2.21 9.15
thr 2.63 10.43
met 2.28 9.21
phe 1.83 9.13
trp 2.38 9.39
asn 2.02 8.80
gln 2.17 9.13
pro 1.99 10.60
asp 2.09 9.82 3.86
glu 2.19 9.67 4.25
his 1.82 9.17 6.00
cys 1.71 10.78 8.33
tyr 2.20 10.07 9.11
lys 2.18 8.95 10.53
arg 2.17 9.04 12.48

There are four structures of proteins. Primary structure is due to the peptide bonds. A peptide bond
forms between the carboxyl group of one amino acid (amino acid 1 in the figure below) and the amino
group of the adjacent amino acid (amino acid 2).


Secondary structure is due to the hydrogen bonds formed between amide nitrogens and carbonyl
oxygens of peptide bonds (e.g. o- helix, | - pleated sheet structures). Tertiary structure is due to
interactions (mostly noncovalent) of side chains (R- groups) of non-neighboring amino acid residues.
Quaternary structure is due to interactions between two or more polypeptide chains in a multisubunit
protein.

Denaturation of proteins:
Denaturation of proteins can be caused by heat, oxidation, reduction, agitation, solvents or x-ray.
Denaturation destroys the natural conformation of a protein. During this process all the bonds are broken
except peptide bonds. Urea and guanidine hydrochloride are denaturating agents widely used for the
preparation of partially unfolded proteins. For the determination of amino acid composition of a
polypeptide, sample is treated with 6 N HCl for about 20-24 hours at 110
o
C and all peptide bonds are
broken (hydrolysis). Partial hydrolysis is carried out by several ways for sequence analysis. For this
pursose the following steps are followed experimentally: (1) Break all Cys-S-S-Cys disulfide bonds by
using -mercaptoethanol; (2) Determine amino acid composition after complete hydrolysis; (3) Identify
N-terminal, C-terminal amino acids of native polypeptide; (4) Break polypeptide into fragments by partial
hydrolysis. Identify N-terminal and C-terminal of fragments. Determine amino acid composition of each
fragment; (5) Determine amino acid sequence of fragments and native polypeptide; (6) Locate S-S- bond
(s) of native polypeptide.


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Specificity of cleavage procedures used in amino acid sequence analysis_______
Procedure Site Specificity _______
I. Terminal cleavages
I.1. Edman degradation C-side of N-terminal Rn=any amino acid
I.2. Carboxypeptidase A N-side of C-terminal Rn=Arg,Lys,Pro
Rn-1=Pro
I.3 Carboxypeptidase B N-side of C-terminal Rn=Arg,Lys,AECys
Rn-1=Pro
II. Internal cleavages
II.1. Cyanogen bromide C-side of Rn Rn=Met
II.2. Trypsin C-side of Rn Rn=Lys,Arg,AECys
Rn+1=Pro
II.3. Chymotrypsin C-side of Rn Rn=Phe,Trp,Tyr,Leu
Rn+1=Pro
II.4. Thermolysin N-side of Rn Rn=Leu, Ile, Phe, Trp, Tyr, Val
Rn-1=Pro
II.5. Pepsin N-side of Rn Rn=Leu, Asp, Glu, Phe, Tyr, Trp
Rn-1=Pro
__________________________________________________________________________

Biological Functions of Proteins are as follows:

(a) Many proteins function as enzymes, the biological catalysts. Enzymes catalyze nearly all reactions
that occur in living organisms.
(b) Some proteins bind other molecules for the purposes of storage and transport.e.g. myoglobin binds O2
in skeletal and cardiac muscle cells, and hemoglobin binds and transports O2 and CO2 in the red blood
cells.
(c) Structural proteins provide mechanical support and shape to cells and hence to tissues and organisms.
(d) Assemblies of proteins can do mechanical work, such as movement of flagella, separation of
chromosomes at mitosis, and contraction of muscles.
(e) Many proteins play a role in decoding information in the cell. Some are involved in translation,
whereas others play a role in regulating gene expression by binding to nucleic acid.
(f) Some proteins are hormones, which regulate biochemical activities in target cells or tissues; other
proteins serve as receptors for hormones.
(g) Some proteins serve other specialized functions. e.g. immunoglobulins, one of the classes of proteins
within the immune system, defend against bacterial an viral infections in vertebrates.

1.5.2. Polysaccharides

Polysaccharides are carbohydrate polymers. Carbohydrates are composed primarily of carbon,
oxygen and hydrogen. This group of biomolecules include simple sugars (monosaccharides), polymers
(polysaccharides) and other sugar derivatives. The most common simple monosaccharides are either
polyhydroxy aldehydes or polyhydroxy ketones.
H CH2OH
| |
C=O C=O
| |
(CHOH)n (CHOH)n
| |
CH2OH CH2OH
Aldose ketose


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Sugars contain either five or six C-atoms. e.g. glucose is a six carbon sugar whereas ribose is a
five carbon sugar. Glucose is a cellular nutrient and is monomeric unit of the storage polysaccharides
glycogen (in animals) and starch (in plants). Cellulose, the structural polysaccharides of plant cell walls,
is also a polymer of glucose. On the other hand, ribose is the carbohydrate component of nucleic acids,
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Disaccharides, meaning "two sugars", are commonly found in nature as sucrose, lactose and
maltose. They are formed by a condensation reaction where one molecule of water condenses or is
released during the joining of two monosaccharides. The type of bond that is formed between the two
sugars is called a glycosidic bond.





1.5.3. Nucleic acids

Nucleotides contain a five carbon carbohydrate (either ribose or deoxyribose), a heterecyclic
nitrogeneous base and at least one phosphate group. The nitrogeneous bases of nucleotides are purines
and pyrimidines. Adenine (A) and guanine (G) are purine bases. Cytosine (C), thymine (T) and uracil (U)
are pyrimidine bases.

Ribonucleotide, monomer of RNA contains ribose, nitrogeneous bases, A, G, C or U and
phosphate whereas deoxyribonucleotide, monomer of DNA contains deoxyribose, nitrogeneous bases A,
G, C or T and phosphate group.

The structure of ribose and deoxyribose is as follows:



Heterocyclic Bases:
Several different bases are found in nucleotides. They are heterocyclic bases or sometimes
referred to as nitrogenous bases because they contain nitrogen within the rings. Some have one ring and
are similar in structure to the compound pyrimidine and, because of that, they are called the pyrimidines
or the pyrimidine bases. There are three of them and they are called cytosine which is found in both DNA

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and RNA, thymine, which is found only in DNA, and uracil, which is found only in RNA. The
abbreviations C, T and U will be used extensively to refer to these compounds.


cytosine (C) thymine (T) uracil (U)

Some of these heterocyclic bases have two rings like the compound purine and, therefore, they are
called the purines or the purine bases. They are adenine and guanine, represented by the letters A and G,
and they are both found in DNA and RNA.


adenine (A) guanine (G)

Nucleosides: If the sugar, ribose is not phosphorylated, the structure is called a nucleoside.



Double Helix structure of DNA

In 1953 James Watson and Francis Crick deduced the three-dimentional structure of DNA from its
x-ray diffraction pattern. The DNA moleculeis a double-stranded, right-handed helix. The important
features of DNA structure:
1.Two helical polynucleotide chains are coiled around a common axis. The chains run in opposite
direction.

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2. The purine and pyrimidine bases are on the inside of the helix, whereas the phosphate and deoxyribose
units are on the outside. The planes of bases are perpendicular to the helix axis, and planes of sugars are
at right angles to those of the bases.
3. Diameter of helix as 20
o
A. Adjacent bases are separated by 3.4
o
A along the helix axis and helix
structure repeated after 10 residues on each chain. So helix structure is repeated at intervals of 34
o
A.
4. The two chains are held together by hydrogen bonds between pairs of bases. Adenine is always paired
with thymine and guanine is always paired with cytosine. The average molecular mass of a nucleotide
pairis about 650. Adenine forms two hydrogen bonds with thymine and guanine forms three hydrogen
bonding with cytosine.
5. The sequence of bases along a polynucleotide chain is not restricted in any way. Each DNA molecule
has its own sequence of bases and the sequence of bases carries the genetic information.
The most important aspect of DNA double helix is the specificity of the pairing of bases. The
pairing of bases is restricted by two factors.
1. Pairing of two purines is not allowed because of steric factor.
2. Pairing of two pyrimidines is not allowed because of hydrogen bonding factor.




Secondary structure of DNA
Secondary structure of RNA

RNA molecules, contrary to DNA, are generally single-stranded. They exhibit irregular, partially
helical structures due to the intrachain H-bonding. The base pairs found in RNA are primarily between
adenine and uracil, and guanine and cytosine.

Forces that stabilize helical structure in DNA and RNA include hydrophobic interactions that
maintain the predominantly nonpolar bases in the interior of the molecule, H-bonds between the bases of
each pair and the so-called stacking energy that results from a combination of van der waals and dipole-
dipole interactions between the bases when they are aligned in paralel planes.

Properties of Nucleic Acids

Nucleic acids denature and renature under appropriate conditions. Nucleic acids unwind or
denature into disordered single strands (random coils) when subjected to high temperature, extremes of
pH or denaturing reagents such as formamide and urea. Denaturation of nucleic acids causes an increase
in their absorption of UV light at wavelenghts near 260 nm. This increase can be followed
spectrophotometrically. The temperature at which DNA is half unfolded is called the melting temperature,
Tm. In case of DNA upon heating there is a sharp increase in absorbance whereas in case of RNA there is

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a small increase in absorbance over a broader range. The temperature which corresponds to the midpoint
of optical density increase caused by heat denaturation is called as melting point and designated by Tm.
Tm for DNA is about 85
o
C. For RNA Tm is is about 75
o
C. Melting point of double helical DNA under
given solvent conditions increases linearly with increasing content of G-C base pairs. So the nucleotide
composition of DNA can be estimated from measurement of Tm. An empirical relationship has been
developed, such as following:

Tm =69.3 + 0.41 (% G + % C) in 0.2 M Na
+

A typical melting curve is as follows:





1.5.4. Lipids

Lipids are water insoluble organic molecules. Some lipids are energy-storage molecules, some are
structural element of membranes, and others are involved in communication within and between cells.
The simplest lipids are fatty acids, which are long-chain hydrocarbons with a carboxylate group at one
end. Fatty acids are most commonly found as part of larger molecules called triacylglycerols (or fats) and
glycerophospholipids. Triacylglycerols, which contain three fatty acid moieties esterified to a molecule of
glycerol are the most abundant type of lipid in mammals.




Glycerophospholipids consists of glycerol 3-phosphate esterified to two fatty acyl group and
usually have a polar group (such as ethanolamine) attached to the phosphate.




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Membrane lipids usually have a polar, hydrophilic (water loving) head and a nonpolar,
hydrophobic (water fearing) tail. In an aqueous medium, such as that found in cells, the hydrophobic tails
of membrane lipids associate, forming two sheets called a lipid bilayer. This structure forms the layer that
is the wall between the inside and outside of the cell.



PROBLEMS

1. What are the major ionic species present at pH 7.5 in 0.15 M solutions of (a) leucine, (b) aspartic acid,
(c) lysine. Use the values given below:
Amino acid pK1 pK2 pKR
Leucine 2.36 9.60 -
Aspartic acid 2.09 9.82 3.86
Lysine 2.18 8.95 10.53

2.Plot the titration curve of leucine with at least 5 points. Use pK values in Problem 1.

3. Estimate (a) pH at which a 50-50 mixture of the +1 and +2 species of lysine would exist, (b) pH at
which the net charge on the molecule is zero, that means pI (c) identify the predominant chemical species
at pH 10.0. Use pK values in Problem 1.

4. To 1 L of a 1 M glycine solution at its isoelectric point is added 0.3 mole HCl. Calculate the pH of the
resultant solution (pK1=2.34, pK2=9.6 for glycine).

5. Describe the preparation of 1 L of 0.1 M histidine buffer, pH 6.5, starting from solid histidine
hydrochloride monohydrate (MW=209.6) and 1 M KOH. PKR=6.0.

6. A native protein is treated with 8 M urea and 1 % |-mercaptoethanol prior to an alkylation reaction
with iodoacetic acid. Write all reactions related with the procedure above with chemical formulas.
7. Ethyleneimine reacts with cystein side chain in proteins to form S-aminoethyl derivatives. The peptide
bonds on the carboxyl side of these modified cystein residues are susceptible to hydrolysis by trypsin.
Why?

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8. Consider the polypeptide whose primary structure is as follows:

Ala-val-lys-leu-phe-arg-cys-tyr

S

S

Glu-met-lys-val-thr-gly-cys-ala
(a) What fragments are produced by digestion with trypsin? (b) What fragments are produced by
digestion with thermolysin? (c) What fragments are produced by digestion with trypsin after reduction
and alkylation of the disulfide bond?
9. Deduce the amino acid sequence of a simple polypeptide from the following results: (a) acid
hydrolysis:Lys,His,Asp,Glu2, Ala,Ile,Val,Tyr. (b) N-terminal analysis with 2,4-dinitrofluorobenzene:
2,4-dinitrophenylaspartate. (c) carboxypeptidase A: Val. (d) trypsin: (Lys,Asp,Glu,Ala,Tyr) and
(Glu,His,Ile,val) (e) N-terminal analysis with 2,4-dinitrofluorobenzene of fragment (Glu,His,Ile,Val): 2,4-
dinitrophenylhistidine (f) thermolysin cleavage of fragment (Glu,His,Ile,Val): (Ile,Glu), (Val), (His) (g)
Chymotrypsin: (Ala,Asp,Tyr) and (Lys,His,Glu2,Ile,Val).
10. Deduce the amino acid sequence of a simple polypeptide from the following results:
(a)Acid hydrolysis: Lys2, Ala2, Phe, Met, Arg, Ser (b) catboxypeptidase B: Lys (c) CNBr: Ser, Met,
Lys and Phe, Lys, Arg, Ala2 (d) Thermolysin: Ala, Phe, Lys and Met, Lys, Ala, Arg, Ser (e) Trypsin:
Lys, Ser and Met, Arg and Phe, Lys, Ala2
11. The -carboxyl group of glutamic acid has a pKa of 4.3. (a) What fraction of these groups will be
unprotonated in a dilute solution of Glu at pH=5.0? (b) at pH=3.8?
12. Which of the following amino acid residues would you expect to find on the inside and which on the
outside of a typical globular protein molecule in solution at pH=7.0?
Thr, Glu, Val, Ile, Asn, Ser, Arg, Phe, Met, Lys
13. A polypeptide can be reduced to yield two peptide subunits whose sequences are:
Chain 1: Ala-Cys-Phe-Pro-Lys-Arg-Trp-Cys-Arg-Arg-Val-Cys
Chain 2: Cys-Tyr-Cys-Phe-Cys
The native polypeptide (intact disulfide bonds) is digested with thermolysin and the following peptides
are obtained. i. Ala, Cys2, Val; ii. Arg, Lys, Phe, Pro; iii. Arg2, Cys2, Trp, Tyr; iv. Cys2, Phe. Indicate
the places of disulfide bonds.
14. An enzyme catalyzed reaction was carried out in a 0.2 M Tris buffer, pH 7.8. As a result of the
reaction, 0.03 mole/L of H
+
was produced.(a) What was the ratio of Tris
+
(conjugate acid)/Tris
o
(conjugate
base)at the start of the reaction? The pKa of Tris is 8.1. (b) What are the concentrations of Tris
+
and Tris
o

at the start of the reaction? (c) What were the concentrations of Tris
o
and Tris
+
at the end of the reaction?
(d) What was the pH at the end of the reaction? (e) What would the final pH be if no buffer were present?
15. How many optical isomers of (a) alanine; (b) threonine are available?
16. The DNA of a deletion mutant of bacteriophage has a counter length of 15 m instead of 17 m.
How many base pairs are missing from this mutant? (Adjacent bases are separated by 3.4
o
A; 1
o
A=10
-10

m)
17. A stretch of double-stranded DNA contains 1000 base pairs, and its base composition is 58 % G+C.
How many thymine residues are in this region of DNA?
18. A pure DNA sample was shown to have a Tm of 85 C. What is the % A+T in this DNA? (Assume the
measurement was made in the presence of 0.2 M Na
+
)