Professional Documents
Culture Documents
Castillo, PhD
Faculty of Pharmacy
University of Santo Tomas
Enzymes
they are high molecular weight proteins which
regulate the biochemical processes in the
organism
Exerts a catalytic effect on chemical reactions, or
they are special organic catalyst.
Biological catalyst a substance that increases
the rate, or velocity of a chemical reaction
without itself being changed in the overall
process.
Enzymes
are special proteins produced only by living cells
which in combination with various non-protein
factors
promote and control with a high degree of
specificity the chemical processes of life.
Their molecular weights vary from about 9000
(hydrogenase) to about 1,000,000.
Characteristics of Enzymes
APOENZYME HOLOENZYME
+ COFACTOR (active)
(inactive)
HCl
Pepsinogen Pepsin
Ex.Lipase,phospholipase,acetycho
linesterase = ex. Of esterases
Stereospecificity- the enzyme in this
case attacks one member of a pair of
stereoisomers which differs only in the
arrangement of atoms, and are
otherwise identical.
a) Temperature an increase in
temperature will speed up enzyme
activity.
Act best at temperatures between 35o & 40oC;
temperatures above 65oC, usually completely
destroy them, whereas their activity is negligible
at 0oC.
Temperature
b) pH there is an optimum pH for
every enzyme.
Enzyme concentration
pH
c) Substrate concentration as the
concentration of substrate molecules
rises, more and more of the active sites
on the enzyme molecules become
occupied.
Once all these sites are busy, the
enzyme is working at maximal capacity,
promote greater production.
d) Enzyme concentration
generally speaking, the amount
of chemical change produced is
directly proportional to the
enzyme concentration.
e) Presence of activators and inhibitors.
Electrophoresis :
to separate individual proteins from one
another
to ascertain the purity of protein
preparation
to estimate the number of components
present in a protein mixture.
This method may also be used to cause a
desire component to migrate away from an
undesired impurity.
Denaturation
The most usual method of separation of
protein & non- protein material
From an extract containing all soluble
components of a plant tissue,
proteins may be removed by heating 70
100oC
and filtration or centrifugation of the
denatured ppt from the soluble low molecular
weight compound.
High concentration of acid may be used
& 5 15% trichloroacetic acid is effective in
precipitating most proteins even at low
temperature.
A tissue may be dried in the oven in which
case the denatured protein remain in the
residue during a subsequent water extraction.
Fractional precipitation w/ concentrations of
neutral salts, generally ammonium sulfate to
separate proteins. Other proteins are ppted only
at higher concentration.
Kohn technique- successive addition of
acetone, alcohol or deoxane to aqueous protein
solution may be used for protein separation.
- This method has been used for preparing many
enzymes & in particular the fractionation of
human blood serum proteins.
Ultracentrifugation
the molecules of proteins in this case are
actually sedimented from solution by application
of very gravitational fields. Since heavier
molecules sediment more rapidly than lighter
molecules.
Other methods of protein separation include:
- absorption of proteins in suitable
absorbents
- partition chromatography on starch or
filter paper.
Enzyme Nomenclature:
1. Name of substrate or group on which the
enzyme acts + suffix ase .
2.Type of reaction involved.
Ex. Oxidases catalyzing oxidation process.
3. Combination of # 1 and # 2.
4. Trivial or empirical names which give no
clues for the enzymes chemical structure. Ex.
Trypsin, steapsin, pepsin, papain etc
Nomenclature & Classification
Units of Enzyme Activity
Enzymes are measured by activity
rather than concentration.
Activity is expressed based on:
1] increase in concentration of one of
the products
2] decrease in concentration of
substrate
3] rate of change in concentration of
coenzyme as a measure of rate
of reaction
maltase
MALTOSE glucose
zymase
GLUCOSE/FRUCTOSE alcohol + CO2
myrosin
SINIGRIN allyl isothiocyanate
Amylolytic Enzymes
Malt Extract - from barley, a dried grain of
one or more varieties of Hordeum vulgare
(Graminae)
- with digestive & nutritive
properties; serves as an aid in digesting
starch
thrombin
Fibrinogen Fibrin
Classification of Proteins:
This classification is based on their products
of hydrolysis, solubilities, precipitation by
salt & coagulation by heat.