intro to bio chem

Biochem -102, 3(2-1)
Elementary Biochemistry

Theory
A general introduction to the science of biochemistry. Ionization of water,
weak acid and weak bases, pH, buffers, diffusion, osmosis and osmatic
pressure. Enzymes: Classification, nomenclature, characteristics,
coenzymes, cofactors and prosthetic groups. Mechanism of enzyme
action. Enzyme inhibition. Carbohydrates: Classification, characteristics,
aerobic and anaerobic oxidation of glucose, biological functions of
carbohydrates. Lipids: Composition and classification, structures of
saturated and unsaturated fatty acids and their properties, characteristics
of fats and oils, general metabolism of fats and oils. Proteins: Composition
and classification, characteristics and classification of amino acids,
peptides and levels of structural organization of proteins, physiological
function and general metabolism of proteins. Nucleic acids: Chemical
composition, structures of DNA and RNA. Functions of DNA and different
types of RNA in the cell.

Introduction to Biochemistry
Chemical Structures, reactions, principles and machanism
behind such interactions with all aspects around Chemistry
Biomolecule’s-Chemical Structures, reactions (Metabolism),
principles and machanism behind such interactions with life in all
its deverse forms and aspects either directly or indirectly
BioChemistry
Molecular Level
“Molecular logic of Life in all its diverse forms can be explained
by Biochemistry”

•Deals with metabolic processes in living tissues a material
called Protoplasm (basis of all life)
•These reactions in Normal way  HEALTHY But
Disorganization SICKNESS/ DEATH
•All components that make life are themselves inanimate but
combinations makes life possible.
•Young emerging science in the 20th century but now a major
Discipline  dependent on the discoveries of braches of
Chemistry (organic, inorganic, physical , analytical etc), Physiology

Scope and importance of
Biochemistry
Now it answers to explanations for the machanisms behind Physiology
Medical sciences  Physiological, Pharmacolgy, Bacteriology,
Pathology
Solutions to clinical problems, remedies to deficiencies like Rickets,
pellegra (B3), Beri-Beri, Scruvy, Aneamia Diagnosis and therapy
Purifying vitamins, hormones, Anti-toxins, vaccines
Enzyme inhibitors, Recombinant DNA technology, genetic engineering ,
cloning , profiling
Mysteries in agriculture, industry, research and all life sciences

Books recommended
Principles of biochemistry by Lehninger (4th edition and onward)
http://www.irb.hr/users/precali/Znanost.o.Moru/Biokemija/Literatura/Lehninger%20Principles%2
0of%20Biochemistry,%20Fourth%20Edition%20-
%20David%20L.%20Nelson,%20Michael%20M.%20Cox.pdf
Medical biochemistry by Mushtaq Ahmed vol-1 edition after 2008
Cell and molecular biology by Gerald Karp 3rd edition and onward
any
http://www.btsdl.cc/cell-and-molecular-biology-by-gerald-karp-6th-edition-tf2432083.html

THE CELL
CELL THEORY;
1. All living organisms are made up of cells and cellular components. May be
uni/multi cellulars.
2. Basic structural and functional unit of Life
3. All cells are produced from preexisting cells.
Properties ;
•A high degree of chemical complexity and microscopic organization.
•Systems for extracting, transforming, and using energy from the environment to do work
•Defined functions for each of an organism‘s components and regulated interactions
•Mechanisms for sensing and responding to alterations in their surroundings.
•A capacity for precise self-replication and self-assembly.
•A capacity to change over time by gradual evolution.

Plasma membranes and cytoplasm
“The outer periphery of the cell that separates its internal contents from the surroundings.”
1. Lipid and protein molecules
2. Thin, tough, pliable, hydrophobic barrier
3. Selectively permeable by Transport proteins
4. Signals by receptor proteins
5. Membrane enzymes participate in reactions
6. Flexible in shape and functions  less strong bondings
7. Can grow as cells multiply
Cytoplasm:
“The internal volume enclosed by the plasma membrane”
1. Aqueous portion cytosol and particular portions gel
2. Rich in enzymes, RNA, metabolites, macromolecules, coenzymes , Ribosomes and
Proteasomes

Movement of materials across
membranes
• Cells surrounded by plasma membraneall
communications through it
• Dual function of membrane 1) must retain the
contents avoid leakage 2) exchange of necessary
materials
• Lipid bilayer is best to protect loss of ions, polar,
amino acids, sugars, hormones etc
• Movements are passively (gradients, no energy
) and actively (energy needed)maintains net
flux (one may exceed other)

Passive transport: Simple diffusion, diffusion
through aqueous protein-linked channel,
facilitated diffusion
• Spontaneous process in which substances move
from a region of higher concentration to low
concentration till equilibrium
• Exergonic  energy from random thermal
motion / collisions
• If substance Electrolyte movement by chemical
gradient or by Electric Potential gradient i.e. by
concentrations or by difference between
charges electrochemical gradients
• E.g. K+ ions across membrane
++++++++++++++++++++++ 3Na
- - - - - - - - - - - - - - - - - - 2K/Cl-

Channels  Move ions nerve impulses,
secretions, muscles contractions, cell volume, open stomata etc
• Integral membrane proteins Downhill
• Always bidirectional till net flux
• Sequence similarities shows this protein
has common ancestry
• Keeps open/close conformations
1. Voltage gated channels: Conformational changes depends on
difference of ionic charges on 2 sides. E.g. K+ channels
2. Ligand-gated: Conformational changes depends on binding
of a specific (ligand) molecule which is not solute itself. E.g.
acetylcholine binds to outer surface to cation channels,
cAMP binds inner to Ca++ channel.
C. Elegans (1000 cells), 90 different genesK+ channels

Non-electrolyte diffuses passively…
• Substance must be in high conc at one of
membrane
• Membrane Permeable to it Solute must cross
aqueous pore without
contact with lipid
Solute must cross lipid
layer by dissolving
It must have polarity match
(NON-POLAR)
Partition coefficient: Ratio of solubility
of solute in non-polar solvent
(octanol/veg.oil) to that in water, when
both solvents are mixed together
2 molecules same P.C, then small uncharged will
penetrate faster. E.g. more CO2,O2,H2O, NO etc
& Less sugars, amino acids, P-compounds
require mechanisms
P.C
Pentrationcm/sec
sugars
caffein
Small values less Greater lipid solubility

Diffusion of water through membrane
best movement example
• Water usually moves rapidly as compared to
other ions / polar solutes selectively
permeable  membrane  called OSMOSIS
“Water moves readily across a semi-
permeable from a region of lower solute
concentration to a region of higher solute
concentration”
Demonstrated by
placing a cell in a non-
permeable solute
solution of different
concentrations across
plasma membranes
Hypertoni
c
/hyperos
motic
solution
Cells shrink
and own H2O
comes out 
cured by gain
of ions
Hypotonic
/hyposmo
tic soltn
Cells swell by
gaining H2O
from outside
 cured by
loss of ions
Isotonic0.85
% NaCl saline
No net flux
Temporary but best example of movement and
shape changes

Uses
• Digestive tract secrete  Liters of of fluid but
reabsorbed by osmosis
 Animals remain in isoosmotic , but in diahhrea occurs if
fails to reabsorb
• Plant cells hypertoniclet H2O inside Turgid (turgor
pressure pushes against cell walls) in hypertonic
solutions plasmolysis
• Aqua porins in some cells more permeable to water
kidney/plant root protein-channels allows this
In congenital nephrogenic diabetes insipidus mutations in
aquaporins vassopressins fail no reabsorbtion higher
urine excretions

Facilitative diffusions: (fast diffusions by
selectively binding membrane spanning proteins that helps in
diffusion)
Solute binds selectively at one end induces
Conformational changes exposing it to other end.
This transporter binds at a side with solute at one
time then to the other side
Similar to enzyme-catalysed reactions;
Specific to even D/L isomers
Obey saturation kinetics i.e. blocked at
saturation
Regulated
Conformational changes are induced (induce fit
model)
Slow rate 100s to 1000s /sec
For Example: GLUT1-5 isoforms, insulin
responsive cells in muscles and adipocytes.
High glucose insulin GLUT4 more
translocated uptake into cells

Active transport  like facilitative diffusion
 transporter proteins  Pumps  moves
selectively solutes  against gradient
V-type pump (only energy changes) & P-
type pump (the pumping cycle &
conformational changes depends on
phosphorylation ) Na-K-ATPase pump
Transportation  SYMPORT or Uniports
e.g. Na+-glucose co-transporter in kidney &
ANTIPORT (species move oppositely) e.g.
Na+/H+ exchanger maintains pH
needs energy  endergonic +
exergonic (metabolic i.e. ATP
hydrolysis, light Absorbance,
electrons transfer etc). E.g. Retina
cells light photons protons
rhodopsin sensation
Examples ;
H+ pumps = membrane of plants 
cytosol pH  growth control
acidification
Ca+2-ATPase pump = outside to in in E.R
H+/K+-ATPases pump= in stomach pump
in H+ and out K+ in exchange

Na-K-ATPase pump tetramers subunits
 transport + maturation & assembly
2/3rd nerve cells energy and 1/3rd
animal cells energy & its electrogenic
(separate charges)2K+ in & 3Na+ out

Properties and functions of water
Polar Molecule of H2O
70% by weight Liquid state due to H-bonds
Between
others
Between
each
other
Like dissolves
like , so polar
solutes and
solvents
dissolves by
interrupting
H20-H2O
interactions
Higher melting,
boiling points and
heat of vaporization,
great internal
cohesion are all due
to H-bonds .
Hydrophilics those that dissolve
easily
Hydrophobics

Ionization of water, weak acids and
bases
Proton hoping
Water is weakly ionizable i.e. few molecules
dissociate although its neutral . Many
properties can also be explained by this.
No free proton exists in
water but forms hydronium
Ionization  Electrical Conductivity and
proton hoping makes it fast (cathode/anode
movements)

To express ionization quantitatively
For a reversible reaction we know;
Equilibrium constant Keq = fixed and
characteristic for any given chemical
reaction at a specified temperature.
It defines the composition of the final
equilibrium mixture
For water;
At 25oC [H2O]=55.5M, for 1L water its
1000/18=55.5
Ionic product
of water
E.C=1 x 10-16 M = Keq

At neutral pH we have same
concentrations of both H+ & OH-
Thus when H+ is high OH- should be
Low. Vice versa
Similarly we also know that;
[H+] [OH-] = 1 x 10-14 M2
Taking –log on Both sides ;
–log[H+] –log [OH-] = –log [1 x 10-14 M2]
Since we know –log [H+]= pH
Thus
pH + pOH = 14At neutral pH, [H+]= 1 x 10-7 M

pH, pOH, pKa
• pH= - log [H+] where [] represents molar concentration
• Strength of H+ in a solution that indicates the measure
of acidic/basic character. Since addition of acids and
bases changes ions concentration in indexes or powers
of 10 and in decimals. Logarithm changes it into an
expressible whole number forms. i.e. 1 x 10-7=[H+]=
pH=7.0=neutrality
• pOH= -log [OH-]
• pKa = - log Ka (defined as that value of pH at which
the amount of weak acid and its conjugate base are
equal) Its helpful in determining the strength of a
buffer as pKa±1=buffer capacity, Ka is the dissociation
constant of an acid. More values stronger acids highly
ionizable.

• The pH of an aqueous solution can
be approximately measured with
various indicator dyes, including
litmus, phenolphthalein, and phenol
red, which undergo
color changes as a proton
dissociates from the dye molecule.
Accurate determinations of pH in
the chemical or glass electrode
method.
• pH is important in clinical sciences
 acidosis/alkalosis.
•

Buffers
“Are solutions or systems that tends to maintain their
own pH when a small amount of acid or base is added
to them”
Chemically two types;
 Acidic = weak acids + conjugate base/salt
For Example, CH3COOH/CH3COO- or CH3COONa acetate
buffer or sodium acetate buffer
• Basic= weak base or its salts
E.G., NH4OH / NH4Cl
Hemoglobin is also a buffering system.

• Buffers serve as first line of defence against any foreign
invader, enzyme reactions, cell biology, storage, switch
on off by maintaining structure-function relationships,
microbiology etc.
• Buffer strength is determined by the Molar
concentrations of the components making it (0.5 M +
0.5 M= 1.0 M)
• pKa= 4.76 , pH=4.8 (max capacity around) of acetate
buffer.
• Among both components weak acids play a significant
role in determining capacity and properties of a Buffer.
• pKa ±1 = Buffer Capacity
Buffers…….

• E.g #2: Sodium Phosphate buffers NaH2PO4/
Na2HPO4/ Na3PO4
• CH3COOH + NaOH  CH3COONa + H2O
• CH3COONa + HCl  CH3COOH + NaCl
• Common ion effect suppressed the dissociation
of acid base which change pH, neutral salt /
water, weak acids as products causing little
changes in pH.
• Titration with 0.1M CH3COOH 10mL with 0.1M
NaOH to understand buffering action of weak
acid
Buffers……. Machanism

Preparation of Buffers
• Prepare a PO4 buffer of pH=7.0 of 250 mL
volume with 0.1 M concentration?
Information is required about Potassium and
Sodium phosphate salts or acid that has pKa
values closest to the required pH.
Information of molar ratios of weak acid and
conjugate is required , from handerson-
hasselbalch equation.
Molar masses required

Selection ;
• H3PO4=pKa=2.1
• NaH2PO4=pKa=6.8
• Na2HPO4=pKa=12.2
pH = pKa + log [A-]/[HA]
7.0 = 6.8 + log [A-]/[HA]
0.2 = log [A-]/[HA]
0.2/1 = log [A-]/[HA]
Taking anti-log on both sides to eliminate
log
Anti-log (0.2) = [A-]/[HA]
1.585 / 1 = [A-]/[HA]
Thus molar ratio of salt (Na2HPO4) is
1.585 and acid is (NaH2PO4) = 1
Total ratios = 1.585 + 1 = 2.585
Volumes required;
Vol of Na2HPO4 = 1.585 / 2.585 x 250 mL
= 153.3 mL
Vol of NaH2PO4 = 1 / 2.585 x 250 mL
= 96.7 mL
Check ; 153.3 mL + 96.7 mL = 250 mL

To get information of mass or weigh of the salt and acids for making buffer;
We need molar mass of
Na2HPO4 . 2 H2O = 178 g
NaH2PO4 . 2 H2O = 156 g
Amount of Na2HPO4 . 2 H2O = 178 g x 153 mL x 0.1 M
1000
= 2.72 g in total 250ml of salt in buffer solution (dH2O)
Amount of NaH2PO4 . 2 H2O = 156 g x 96.7 mL x 0.1 M
1000
= 1.508 g in total 250ml of acid in buffer solution (dH2O)
Thus first take 200 mL of dH2O and dissolve Na2HPO4 . 2 H2O = 2.72 g and
NaH2PO4 . 2 H2O = 1.508 g and make volume upto 250 mL
Check pH
Check % error

Problem # 2.
Prepare a buffer solution of Na-
Acetate of pH=5.76 , 0.1M of 1 litre
volume. (pKa=4.76 of CH3COOH)

Solution
5.76 = 4.76 + log [A-]/[HA]
1 = log [A-]/[HA]
log
10/1 = [A-]/[HA]
Total molar ratios = 10 + 1 = 11
Volume of acid = 1/11 x 1000 mL
= 90.9 mL
Volume of salt = 10/11 x 1000 mL
= 909.1 mL
Selection= CH3COOH/ CH3COONa
CH3COOH specific gravity 1.052 g/mL,
99%
Amount of CH3COOH = 60 g x 90.9 x 0.1 M
1000
= 0.545 g in total 1000ml of salt in buffer
solution (dH2O)
Amount of CH3COONa= 82 g x 909.1 x 0.1 M
1000
= 7.5 g in total 1000 ml of acid in buffer
solution (dH2O)
NOTE= FOR SOLID SALT CH3COONa WE CAN
WEIGH BY WEIGHING BALANCE BUT FOR
LIQUIDS LIKE CH3COOH WE NEED TO USE
VOLUMES EQUIVALENT TO MASS
CALCULATED
(Info about purity and sp.gravity will help
here)

We know CH3COOH specific gravity= 1.052 g/mL, 99%
that is
1.052 g = 1 mL
1 g = 1/1.052
5.45g = 1/1.052 x 5.45
= 5.18 mL
so 99% pure acid required in vol= 5.18 mL
1% = 5.18/99
100% = 5.18/99 x 100
= 5.3 mL of 99% pure is
required to make a buffer
check pH , calculate % Error, check individually while adding
components which component plays a vital role in pH.

Numerical assignment (hand written)
• Calculate the concentration of H+ ions in a solution of pH=0.5 ?
• Calculate the OH- ions concentration in a given solution of pH 1.1?
• Calculate the number of molecules of glucose present in 360 grams?
• Calculate the number molecules of H2SO4 in 1 mL if its of 1.84 specific gravity?
• What will be the molar volumes of NaH2PO4 and Na2HPO4 required to make a
buffer of 0.5M , if their amounts are 1.25 g and 1 g respectively used for
preparing buffer.
• Calculate the amounts of acetic acid and sodium acetate, to prepare pH=7 buffer,
of 0.25M and 175mL volume?pKa of acetic acid= 4.76, 96% purity of acid.
• What will be the pH of a solution containing OH- ions 1.34 x 10-4 ?
• What will be the Ka of an acid with pKa value close to 4.8?

What are enzymes ?  Biological catalyst
Enzymos  some catalytic agent derived from
Yeast
Catalyst is an agent that accelerates rate of the
reaction Vo by lowering the Ea (energy of activation)
without itself appearing in the products.
Ea  the energy in Cals/mol required to be
supplied to the reaction to initiate.
Both Vo and Ea are inversely proportional to each
other.
Moles of product formed per unit time is rate of
the reaction
Specificity  Absolute (1 En 1 S )and relative (1
En (range of substrates)  (gluco/hexokinases)
Mode of actions  lock and key mechanism &
induced fit model

Factors affecting enzyme’s activity or Vo
1. Enzyme concentration: [E] α rate of reaction Vo
• 1st order reaction  with increase of one reacting
species or factor there is proportional increase or
decrease in rate of the reaction
• Depending on hormones, metabolites, other
factors increase or decrease [E] i.e. rate of
synthesis and degradation (in hours/days)

Factors
2. Substrate concentration:
those following Machelis-menton kinetics
start [S] increases the Vo proportionally 1st
order reaction when substrate is less and
before Km value.

 Effect of temperature: within limited range i.e. due to denaturation of proteins of
enzymes, temperature increase increases Vo (Q10 principle, 30oC valid)
• Since temperature α K.E α Vo collisions of reactants
• But maximum activity seen at each enzymes’ optimum temperature.
• Plants tolerate max at 60oC and humans 37oC
 Effects of pH: within limited range of pH since pH changes the ionic states of proteins
which keep them intact so certain ionic state of enzyme protein  structure + function
(active site)
• But maximum activity seen at each enzyme’s optimum pH.
• Trypsin = 8-9 pH, salivary amylase= 6.4-6.9
 Effects of products: A+B  C+D (if reversible)
• Reaction may proceed more faster if products removed or reactant increased
• Reaction rate may slow if products has similarities with substrate
 Cofactors and inhibitors: cofactors (bridge S active site) and coenymes are required to
make enzyme complete and to increase rate of the reaction e.g. Fe++(cytochrome oxidase,
catalase etc), Cu++(cytochrome oxidase), Zn++ (alc. Dehydrogenase, carbonic anhydrase),
Mg++(hexokinase, pyruvate kinases etc), Mn++ (arginase), K+ (pyruvate kinases), Ni+
(urease)
• Organic or metalloorganic (group transferring)  NAD, FAD, FMN, CoASH, etc
coenzymes
• Apoenzyme (protein part) cofactors (tightly bound, prosthetic groups) holoenzymes
• Inhibitors interfere

CLASSIFICATION OF ENZYMES
OTHLIL

Classification
HEXOKINASES = 2.7.1.1
(ATP; D-Glucose-6-Phosphate
Transferases
“Ase” after substrate
Lipase, urease, proteases
Machanism = transmethylase,
oxidases etc
Trivial names= No
relationship but may be latin
etc.
Pepsin, trypsin, chymotrypsin
IUBMB
4-digits + systemic name + units
i.u.= 1 umole [S][P]/min at 30oC at opt
pH.
Katal = mole of substrate without 30oC
More than 2 names = succinyl-coA synthatase
OR succinate thiokinases

Types of inhibitions of Enzymes
Reversible inhibitions
IRReversible inhibitions
IAAThiols
DIPF Acetylcholine esterases
Heavy metals (Ag+) EnZ-SH
(Mercaptides)
Ampicillin  transpeptidases (C.W)

Competitive enzyme inhibitions
When resemblance in structure between the
[I] and [S]
•Binds to some or all free enzymes
•Km and Vmax changes
•Lowers the [ES] complex
•Can be reversed by increasing [S]
 Important as metabolic antagonists,
chemotherapy against bacterial, viral and
malignant cancer cells
For example;
Succinate dehydrogenase
tRNA (puromycin), glutamine(azaserine), Met (ethionine)

In cell wall synthesis and DNA
synthesis and stops cells from dividing

•No resemblance with substrate
•Inhibitor doesn’t binds the active site but
sites other than active site.
•Binds to both [E] and [ES]
•Causes change in the 3-D structure of
active sites
•No effect of increasing [S]
For examples ;
1. Ions of heavy metals
2. EDTA chelates Ca++
3. Fluoride removes Mg++

Binds to only [ES] complex.
Makes [ESI] complexes
Not reversed by adding excess [S]
but may be by changes like pH,
temperature, etc
For example; Where two or more
substrates are required like DNA
polymerases being inhibited by
ddNTPs

Isozymes / iso-enzymes
Enzymes that catalyze same reaction (same
organism) but are Physically & Chemically distinct
as produced at different locations.
Lactate dehydrogenase (LDH I-V) Creatine Kinase (CK 1-3)
Each 34,000 Mol. Wt on
electrophoresis

Chemically distinct
LDH
2 genes seperately
encoding 2 polypeptides
(H & M types)
Combines into a 4-
polypeptides unit
LDH-1= H4 (heart), LDH-
5=M4 (skeletal muscles)
CK/CPK
2 polypeptides
B & M
Combines into Dimer
CK-1= BB (Brain), CK-
2=MB (Heart
myocardium), CK-3= MM
LDH-2 & CK-2= Myocardial Infarction
in serum appears (DIAGNOSIS)
Lungs,
kidney,
heart
,liver etc
Brain,
gonads,
heart ,retina,
muscles etc

Ribozymes
“Certain RNA molecules have catalytic properties
similar to Enzyme therefore called ribozymes”
Substrates Others RNAs or part
of ribozymes itself
Therefore believed to be first gene first enzyme evolutionary. As
in HIV viruses reverse transcriptasescDNA

Examples of ribozymes
1.414 bp RNA in venomes of tetra himena
catalyses self elongation from host nucleotides
2.M1-RNA in Rnase-Pprecursor t-RNA cleavge
3.RNA in self splicing of spliceosomemRNA
4.Small RNA viruses of plants
5.RNA part of rRNA in Ribosome
peptidyltransferase activity
Iron-protoporphyrin-rings  heme  Catalase
H2O2H2O + O2
Benzidine test (Blood detection)

Proteins are complex organic
nitrogenous compounds made up of
building blocks called amino acids
linked by a covalent linkage called
peptide bond
>300 amino acids known  20 participate in Protein
synthesis standard/primary/normal amino acids (encoded
by gene/codons)
 Proteogenics22Pyrolysine (methanogenics) &
Selenocysteine (25 in Eukaryotes and prokaryotes)
9 Essential amino acids & 11  non-essential

Non-standard/ secondary amino acids
Not in proteins usually but have essential metabolic and
physiological roles
1) Pentothenic amino acidVit-B5 Co-enzyme A
2) GABAgamma amino butyric acidNeurotransmitter
α-Carbonα-COOH & α-NH2 19-α- & 1 – Beta amino acid=proline

Uses and types
1. Plastics
2. Drugs
3. Chiral catalysts
4. Food supplements
Aliphatics, acyclic, aromatic (tyrosine=UV
absorbance)
Some are non-polar (Hydrophobic
clusters)= glycine, methionine, proline
etc
Some are polar (uncharged)= serine,
asparaginine, etc
Some are polar (charged)= Aspartic acid
as –VE , Lysine as +VE

Peptide bond
DIPEPTIDE
If α-COOH of 1st
amino acid
condenses with the
α-NH2 of other
adjacent Amino
acid Acid-amide
bond (Peptide)
• Amino acid residues
• Unusual peptide bond
(GSH=γ-glu-cys-gly)
• Polypetides (>10 a.a)
• Proteins (M.W>10,000)
 H-Bonding by (-CO-NH-)

Classification of proteins
1. Simple proteins
2. Conjugated
3. Derived proteins
Based on
function
Based on
axial ratios
Fibrous (long thread
like) proteins e.g.
collagen, keratin
Globular (spheroid)
proteins e.g. albumins,IG

Based on functions
1.Catalytic proteins
2.Regulatory
3.Structural
4.Transport
5.Immune proteins
6.Contractile
7.Genetic
8.Storage
Enzymes
Hormones (Insulin, GH)
Collagen, Keratin
Transferrin, albumin
Antibodies, IGg
Actin & Myosin
Histones , ssDBP
Casein = Milk
Ovalbumin= Egg

3. Derived proteins
Primary derived proteins Secondary derived proteins
When some / all cross linkages
that holds the protein molecular
structure intact are cleaved
pH,Temperature, ultrasonics,
shake vigorously, alcohols
etc
Intermediates of
progressive hydrolysis of
proteins by enzymes/acid
Proteins Proteoses Peptones
Polypeptidesoligopeptides
Dipeptide Amino
acids

Simple Proteins
Made up amino acids and its derivatives
only i.e. these are hydrolysis end products
1. ALBUMINS 2. GLOBULINS
Serum albumin,
Ovalbumin, legumelin,
lactalbumin etc
Serum Globulin,
Ovoglobulin, legumin,
lactoglobulin, myosin.

Examples
3. Globins 4.
Histones
5. Collagen
Histidine residues
not basic
Slightly basic pH
Heme is Same in all
but globins
differenthemoglob
in
Proline , glycine
hydroxy-proline,
cysteines
Resistant
Keratin = shell,
hoofs
Elastin=arteries,te
ndon
Rich in basic
proteins like
arginine and
lysine
+ve charge
nucleic acids
–Ve
chargenucleo
some

Conjugated proteins
Made up amino acids and its derivatives
covalently linked to a non protein part
called prosthetic group
Glycoprotei
ns
Chromoproteins Metallopro
teins
Phosphoproteins
Lipoproteins
Nucleoproteins

Practical
• Determination of pH value of biological fluids.
Preparation of buffers of definite pH.
Estimation of optical activity by polarimetry.
Qualitative analysis of carbohydrates.
Qualitative analysis of urine for normal and
abnormal constituents - albumin, acetone
bodies and sugar. Estimation of glucose in
biological fluids. Determination of acid,
sponification and iodine values of fats/oils.
Estimation of lactose and casein in milk.

Outlines to cover
• Preparation of solutions, normal,
molar, ppm, percentage, dilutions,
determination of pH of solution,
buffer preparation,
Concentrations, Molarities,
pH, pOH

Determination of protein and oil
contents
• Biochemical tests for proteins and amino acids
• Biochemical tests for oils and fats
Estimation of glucose , fructose and starch
(Carbohydrates biochemical tests)
• Molisch test, iodine test, barfoed, benedicts ,
salivanoff tests , phenyl hydrazine test etc
• Estimation of lactose and casein in milk

Determination of optical activities of
sugar solutions
• Saponification and I2 values of
fats and oils
Determination of acetone and
albumin in body solutions

Basics to start
• Milligrams (mg)= 1000th part of a gram 10-3 g or
0.001 grams
• Kilograms = 103 grams or 1000 g
• Parts per million (ppm)= measures smallest levels of
pollutants in air, water, body fluids. i.e. mass of a
pollutant and a solution
Ppm= 1,000,000 Mc/Ms (Mc = mass of component,
Ms= mass of solution)
E.G. = 1 mg/ Kg as mass per unit of mass

Ppm……
• E.g # 2; mg / Lit as Mass per volume
• Parts per billions (ppb)= 10-9
• Parts per trillions (ppt)= 10-12
• Parts per quadrillions (ppq)=10-15
1 L ≡ 1 dm3 ≡ 1000 cm3
1 L ≡ 0.001 m3 ≡ 1000 cm3 1 m3 =1000 L
• Distilled water d H20, dd H20, ddd H20
(Distilled and deionized)

Molarity (M), Normality(N), molality(m)
• Molarity: Number of Moles of solute dissolved in
one Litre (1 dm3 =1000cm3) of solution.
Unit=moles/ dm3
M=n/L . [Mole = given mass/ molar mass]
• Molality: Number of Moles of solute dissolved in
one Kilogram of solution.
• Normality: Number of gram equivalents of solute
dissolved in one Liter of solution.

• 1 mole of Glucose = 180.2 g of glucose
• Molar mass / # of replaceable H+ or OH- ions
OR (Gram equivalent weight)
Molar mass / valence number
• 1 gram equivalent of H2SO4 = 98/2 = 49 g= 1 g.eq
So 49 g of H2SO4 dissolved in 1 Liter= 1.0 N H2SO4
Also ; Ca(OH)2= 74/2 = 37 g = 1g.eq/Lit = 1 N
solution
• Avogadro's number = 6.022 x 1023
atoms/molecules in a mole

• Bases = Proton Acceptor e.g. a monoacidic
base NaOH ↔ Na+ + OH-
• Acids = proton donor e.g. a monobasic acid
HCl ↔ H+ + Cl-
• Buffer = solutions or systems that maintain
their own pH when a small amount of acid or
base is added to them. E. g.
NaH2PO4/Na2HPO4 sodium phosphate buffer

Solute , solvent , solution and mixture
• Solute : Segment of a solution that is in lesser amount and determines
the strength of a solution. Its is always dissoolved in other substances. E.g.
solid, liquid, gas
• Solvent : Segment of a solution that is in greater amount and
accomodates a solute usually. E. g. Gas or liquids
• Solution: A homogenous mixture of 2 or more substances either solids,
liquids or gases or combinations of both. Some interactions or linkages do
develop, hard to see through when soluble E.g. soft drinks, 10 % salt
solution
• Mixture: in mixtures components cannot dissolve but are combinations
of each other. Solution is a mixture but mixture cant be a solution. Can be
easily separated by physical means like heating , filtration etc. No
interactions exist e.g. sand mixed with salts.

Precision and accuracy and specific
gravity
• Precision = degree to which an instrument or process
repeats the same value or How much reproducible the
experiment is.
• Accuracy = degree of closeness to true values
• E.g. refrigrator set at 38 degrees gives
38.1, 38.5 , 38.7, 38, 38.5, 38.9, 39 etc (More accuracy
less precise)
• Specific gravity= weight of substance in 1 mL of
solvent/solution e.g. 1.84 g/mL H2SO4
Ratio of density of substance/density of refernce substanc

• Absorbance = bulk phenomenon, homogenous or
uniform, as a fluid or gas dissolves in a liquid or
solid absorbant distributes thoroughly. E.g. ink
drop in water glass
• Adsorbance = surface based phenomenon,
higher at surfaces not homogenous, at start
increases but then become slow at equillibrium ,
adheres only physically. E.g. gasses trapped in the
spaces of stars

%composition, analyte, titration,
standard solution, blank, sample,
 w/v, w/w, v/v.
 The target to qualify or measure in a mixture
 Volumetric method of determining the concentration of an
unknown solution whose volume is known by the help of a
standard. Sometimes indicators help. E.g. acid-base
titrations, redox-titrations etc
 Whose concentration is known
 Contains everything present in a reaction mixture except
your target analyte, gives zero readings of an experiment.
 Sample is the mixture containing unknown amounts of
your analyte and its analysis is to be done. It is always a
crude.

Ionization of water, weak acids and
bases
Proton hoping
Water is weakly ionizable i.e. few molecules
dissociate although its neutral . Many
properties can also be expalined by this.
No free proton exists in
water but forms hydronium
Ionization  Electrical Conductivity and
proton hoping makes it fast (cathode/anode
movements)

To express ionization quantitatively
For a reversible reaction we know;
Equilibrium constant Keq= fixed and
characteristic for any given chemical
reaction at a specified temperature.
It defines the composition of the final
equilibrium mixture
For water;
At 25oC [H2O]=55.5M, for 1L water its
1000/18=55.5
Ionic product
of water
E.C=1.8 x 10-16 M = Keq

At neutral pH we have same
concentrations of both H+ & OH-
Thus when H+ is high OH- should be
Low. Vice versa
Similarly we also know that;
[H+] [OH-] = 1 x 10-14 M2
Taking –log on Both sides ;
–log[H+] –log [OH-] = –log [1 x 10-14 M2]
Since we know –log [H+]= pH
Thus
pH + pOH = 14
At neutral pH, [H+]= 1 x 10-7 M
Similarly –log [OH-]= pOH

pH, pOH, pKa
• pH= - log [H+] where [] represents molar conc
• Strength of H+ in a solution that indicates the measure
of acidic/basic character. Since addition of acids and
bases changes ions concentration in indexes or powers
of 10 and in decimals. Logarithm changes it into an
expressable whole number forms. i.e. 1 x 10-7=[H+]=
pH=7.0=neutrality
• pOH= -log [OH-]
• pKa = - log Ka (defined as that value of pH at which
the amount of weak acid and its conjugate base are
equal) Its helpful in determining the strength of a
buffer as pKa±1=buffer capacity, Ka is the dissociation
constant of an acid. More values stronger acids highly
ionizable.

Buffers
“Are solutions or systems that tends to maintain their
own pH when a small amount of acid or base is added
to them”
Chemically two types;
 Acidic = weak acids + conjugate base/salt
For Example, CH3COOH/CH3COO- or CH3COONa
acetate buffer or sod.acetate buffer
• Basic= weak base or its salts
E.G., NH4OH / NH4Cl
Hemoglobin is also a buffering system.

General Lab safety procedures
• Students are allowed in the laboratory only in the
presence of a tutor.
• Before entering the laboratory, you need to wear a
laboratory coat and soft shoes (closed).
• In the laboratory, you need to work carefully, avoid
unnecessary conversations and keep your workplace
clean.
• Please read the warning signs and symbols placed on
the reagents prepare an assignments.
• Preparations and reagents must not be examined by
taste.

• Eating and drinking are not allowed in the laboratory.
• Use distilled water, electricity and gas efficiently.
• Take particular caution when handling concentrated acids,
bases, poisons and flammable liquids. Concentrated acids,
bases and poisons should only be collected by dipping the
pipette. Used acids and bases should be poured out into
the sink in such a manner as to avoid burns caused by
drops of liquid deflected from the wall of the sink (while
pouring, hold the mouth of the vessel as close as possible
to the drain, gently rinse with water).
• Flammable liquids should be used on premises without
ignited burners or other sources of open flames. They
should be stored in tightly sealed vessels.
• Cover your wounds and cuts.
• Label each container and keep records like lab books.

• The gas installation should be used with caution. Unnecessary burners
should be immediately turned off. When wash with alcohols always dry
before coming close to falmes.
• In the case of burns to the skin, mouth or eyes, immediately wash the
corrosive liquid with plenty of tap water and notify the tutor. Then,
neutralize the acids with 5% sodium bicarbonate, and the bases with 1%
acetic acid. Compounds used for neutralization can be found in each
laboratory.
• Avoid woolen wears, long hairs opened, nails cutted.
• Be-aware of using all instruments and chemicals by reading SOPs and
MSDS.
• Place hazardous and flammable liquids and bottles at a bottom space
below your standing position. Any instance should be informed to the
supervisor first.
• Disposals and disposal areas should be defined. Avoid sharp edges.
• Before leaving the lab, the workplace, reagents and equipment should be
put in order. Wash the lab glass. Close the gas valves. Turn off the taps.

MSDS (material safety data sheet) of
different chemicals like HCl, ethanol
etc
SOPs to follow like  pH meter etc
Safety measures for  radiation,
chemical, biological hazards etc
(a manual is available in USC health and safety dept website)

Solution
5.76 = 4.76 + log [A-]/[HA]
1 = log [A-]/[HA]
log
10/1 = [A-]/[HA]
Total molar ratios = 10 + 1 = 11
Volume of acid = 1/11 x 1000 mL
= 90.9 mL
Volume of salt = 10/11 x 1000 mL
= 909.1 mL
Selection= CH3COOH/ CH3COONa
CH3COOH specific gravity 1.052 g/mL,
99%
Amount of CH3COOH = 60 g x 90.9 x 0.1 M
1000
= 0.545 g in total 1000ml of acid in buffer
solution (dH2O)
Amount of CH3COONa= 82 g x 909.1 x 0.1 M
1000
= 7.5 g in total 1000 ml of salt in buffer
solution (dH2O)
NOTE= FOR SOLID SALT CH3COONa WE CAN
WEIGH BY WEIGHING BALANCE BUT FOR
LIQUIDS LIKE CH3COOH WE NEED TO USE
VOLUMES EQUIVALENT TO MASS
CALCULATED
(Info about purity and sp.gravity will help
here)

pH metry
Use of a pH meter  2 electrodes 1 cm apart  calomel electrode (Hg / HgCl2 amalgum)
Reference electrode (filled with 0.1 M HCl ) for comparison purpose with the given
solution
Working;
Given solution  certain amount of H+ ions as compared to 0.1 M HCl  potential
difference  e.m.f (Electr Motive Force)  electrons flow  galvanometer reads
1. Check pH of water
2. Add acid into separate beaker of dH2O and then salt of conjugate base into separate
beaker
3. Check both of the beaker’s pH values (wash before and after always) and note which
one is more close to our required pH, this component is the acid which contributes in
pH determination more
4. Now add 1 mL 1M NaOH in each of the two separate beakers and check the pH
changes of addition of strong base, note it will be the acid component that will resist
maximum and is the component which contributes in buffer strength maximum.
5. Now mix both the component’s solutions and note the pH again , it will be closest to
our required now when both components are present , check % error if any.

To prepare N/10 250 mL of H2SO4
solution?
Concepts of molarity , normalty, solute , solvent, solution, standard solution , primary
and secondary standard solutions are important alongwith titrations and indicators etc
Preparation of a secondary standard solution and then standardization by titration with
o.1N Na2CO3 solution alongwith indicator to confirm what exact is the concentrations
made.
0.5g/100mL dH2O or 0.1% in alc,
Acidic= reddish orange <3.1
Basic= Yellow >4.4
Mutagenic , methyl orange

protocol
1 ml H2SO4 in 100 mL dH2O
Add it in a burrette for titration and detetrmination of its normality
In a flask add 5 mL of 0.1 N Na2CO3 (seperately prepared by molar mass  100mL)
Add a drop of methyl orange in it.
Dropwise add H2SO4 from burrette till end point appears from yellow to pinkinsh
orange or reddish
Repeat 3 times
Note volume consumed to titrate and complete reaction
Use N1V1=N2V2
Once normality known calculate volume of dH2O required to dilute to attain
desired normality

Calculations
Unknown H2SO4 : N/10 Na2CO3 (Known)
N1V1 : N2V2
N1(?) x 3.53 mL(Supposed) = 0.1 N x 5 mL
N1= 0.14N (supposed)
Unknown volume H2SO4 : N/10 H2SO4 (Known)
N3V3 : N4V4
0.14 x (??) V3 = 0.1 N x 250 mL
V3= 178.6 mL of H2so4 solution
Volume of dH2O = 250- 178.5 = 71.4 mL

Qualitative analysis of carbohydrates
in the given solution(2-5%)
• Molisch’s test (group identification test for carbohydrates)
• Iodine test (polysaccharides like blue for starch)
• Ammonium sulphate test (dextrin in polysaccharides purple)
• Barfoed’s test (mono saccharides from di-)
• Benedict’s test (reducing sugars like monosaccharides)
• Fehling’s test (for water soluble ketones and differ from
aldehydes)
• Pholoroglucinol test (pentoses like ribose)
• Seliwanoff’s test (aldoses and ketoses)
• Phenyl Hydrazine test (reducing sugars form osazones crystals
shape specific for type of sugars maltose give sunflower shape)
• Hydrolysis of Sucrose and Starch (acid / enzymatic hydrolysis)
• Physical state , solubility , taste also gives etc.

Furfural formation
(for colored end products)
• Sugars treated  strong acids + heated 
dehydration and hydrolysis  forms specific
intermingled structures  FURFURALs 
depending on furfural reacts with specific
coloring compounds colored complex
• For example;
C5H10O5  C5H4O2 (Furfural)-3H2O
H2SO4

Molisch test (group test)
• For free & conjugated carbohydrates(glycoproteins)
• Reagent contains = 1g alpha naphthol (in EtOH) + H2SO4
(forms furfural and condenses with alpha naphthol)
• Procedure;
 3 mL G.S  test tube  2-3 drops of molisch reagent
 mix  3 mL conc H2SO4 from sides rotate but
don’t shake  presence of hydrolysed reddish violet
ring at the junction of 2 solutions  carbohydrates
present

Iodine test (polysaccharides)
• Reagents = I2 + KI (self oxidizing by atmospheric/inhibitor) +
HCl (acids for oxidation) adsorption
• Adsorption reversed by alkali
• Adsorption occurs in larger molecules at least 8-Carbon
chain
• Sometimes heating also reverses adsorption
Procedure: 3 ml G.S+ 1 drop conc.HCl+1 drop I2 solution.
Results: starch  Blue(amylose), Dextrin  reddish purple,
Glycogen (amylopectin like) reddish brown, no color  no
polysaccharides

Benedict’s test (for reducing / non)
semi-quantitative
• Free aldo/keto + some disacharides reduces
metals like CU+2CU+
• Sodium carbonate + sod.citrate (avoids Cu ppt)+
CuSO4
CuSO4  Cu++ + SO4
Cu+2 + HOH  CU(OH)2 BLUE
CU(OH)2  CU+2 + 2OH-
CU+ + amount of reducing sugar (CH2O) CU2O
(red ppt) cupric oxide
Detects glucose in urine in clinical labs

Benedict test
• 5 ml benedicts reagent + 8 drops of G.S + mix
+ boil 2 min + cool
• Results :
Green  0.1-0.5 %
Yellow  0.5 – 1.0%
Orange  1.0 – 1.5 %
Red  1.5- 2 %
Brick red  >2%

Barfoed’s test (differ mono/disach)
• Reagents : Cu-acetate and glacial acetic acid
(weak)
• Principle: its also reduction metals like
CU+2CU+ in weak acid presence (acids
promote oxidation not reduction)mono
being strong reducers gives fast reduction
even environment not favourable and di gives
slow reduction  time dependent

Barfoed’s test
• Procedure :
• 2 ml barfoeds reagent + 2 ml G.S + mix + note
time + start boiling in water bath
• If red ppt / reddish orange in 2-5 mins boiling
+ cool  monosaccharide
• If ppt after 5-15mins boil  disaccharides

Salivanoff’s test (+ve for ketose)
• Also called resorcinol test (coloring agent in
reagent mix for furfural reaction) along with HCl
• Principle: Carbohydrates+ HCl  furfural  only
ketohexose + resorcinol  cherry red colored
complex  +ve for ketohexose (Fructose/
sucrose) glucose will be faint pink
• Procedure: 3 ml salivanof reagent + 1 ml G.S +
mix + boil for 30 – 60 secs  cherry red / dark
pink (ketohexose)

Osazone / Phenyl hyrazine test (specific for
many reducing mono-disaccharides)
• Reagents : phenyl hydrazine+ Na-acetate (1:2w/w)
• Glucose etc  hydrolysed  C6H5NH2  +ve result 
Glucosazone (bundle grass needle crystals)+ NH3 + H2O
• Procedure: 5 ml G.S + 0.3g osazone mix + 3 drops gl. Acetic
acid + boil + cool dry on filter paper  observe under
microscope
• Results : yellow bundle grass shaped (glu/fruc), galactose
(needle shaped/flufy), maltose (petals sunflower), lactose
(puff ball)

intro to bio chem

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