Download Qualitative determination of Amylase activity

CLS 331 •
Experiment •
No.1
The hydrolytic activity of salivary
amylase on starch
Introduction
Amylase is a hydrolytic enzyme which breaks down many
polysaccharides such as starch.
Starch
Is a polymer of D- glucose units linked by α-1, 4 glycosidic
bonds.
The starch is made up of two polysaccharides:
Amylopectin (branched-chain polysaccharide)..
Amylose (unbranched-chain polysaccharide
The hydrolytic effect of amylase on starch results in yielding
maltose (composed of two D-glucose molecules ) as the
end product.
There are two broad groups of amylases: α and β- amylases.
The β- amylases rapidly hydrolyse the amylose portion of
starch to maltose by acting on residues at the non reducing
terminals.
They hydrolyse α-1, 4 glycosidic links in polysaccharides
so as to remove successive maltose units from the nonreducing ends of the chains.
The α amylases, in contrast to the β- amylases, cause a rapid
loss of the capacity of amylase to give a blue colour with
iodine, also the rate of appearance of maltose is much
slower in the α amylases catalysed reaction than in the βamylases catalysed one.
Types of amylase:
α - amylases – human amylase
β- amylases – bacteria and plant amylase
Source of amylase:
1- Pancreas
2- Salivary gland
Secretion:
Serum or urine
Hyperamylasemia
Increase the secretion of amylase in blood
Some Causes of Hyperamylasemia
Acute pancreatitis, Salivary gland lesions, etc
The Assay
In any enzyme assay, the rate of the reaction can be
known by measuring the amount of substrate (s) that is
utilized or the amount of product (s) that is formed in unit
time, it also called enzyme unit.
In the case of amylase,
The substrate is starch (colourless)
The product is maltose (colourless)
They can be converted to coloured products by specific
chemical reactions.
Starch +Amylase
Starch + Amylase + Iodine
(qualitative)
maltose (colourless)
blue colour
Principle
At a pH of about 6-7 and in the presence of chloride
ions, α amylases catalyses the hydrolysis of starch to
maltose with the intermediate formation of various
dextrins.
Dextrins are polysaccharide produced by the hydrolysis
of starch
Starch + Cl+ + α amylases
maltose + intermediate
formation of dextrins
Steps of the reaction:
1. Starch + α amylase + Higher dextrins
2. Intermediated dextrins
3. Lower dextrins + maltose
Iodine
Iodine
Iodine
blue colour
reddish brown colour
colourless
This point known as Achromic point ( time taken to reach the
point at which the reaction mixture no longer gives a colour
with iodine)
Procedure
Part1:
ml saliva
Control 1
Test1
Stock saliva
9.5ml H2O
0.5ml saliva
5ml
Test tube1: mix then incubate at 37 C
Control tube1: gently boiled for 2 min, cooled and then
placed in the water bath at 37 C
Part 2
Control 2
Test 2
5ml starch
2ml phosphate buffer
1ml NaCl
Mix well, then incubate at 37⁰C X 5-10 min
After incubation,
C1
T1
1 ml
1 ml
C2
In incubator
T2
Spotting Plate
Add 1 drop of iodine solution in 2 rows
C
Add 3-4 drops from C2
Add 3-4 drops from C2
after 1 min, mix
Add 3-4 drops from C2
after 1 min or half min, mix
Add 3-4 drops from C2
after 1 min or half min, mix
add 3-4 drops from C2
after 1 min or half min, mix
T
Add 3-4 drops from T2
Add 3-4 drops from T2
after 1 min min, mix
Add 3-4 drops from T2
after 1 min or half min,
mix
3-4 drops from T2
After 1 min or half min,
mix
3-4 drops from T2
After 1 min or half min,
mix