PRACTICAL BIOCHEMISTRY II

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RESPIRATION MODULE
 PRACTICAL BIOCHEMISTRY II
-RESPIRATION MODULE-

1) MEASUREMENT OF Hb LEVEL
(Cyanmet Hb)

2) MEASUREMENT OF LIPID
PEROXIDES IN THE
HEMOLYSATE
 1. MEASUREMENT OF Hb LEVEL
BY CYANMETHb METHOD

OBJECTIVES

MEASUREMENT OF Hb LEVEL IN BLOOD BY

QUANTITATIVE CYANMETHEMOGLOBIN
METHOD
 Principle

In this method all of Hb derivates is changed to

cyanmetHb that is more stabil after addition of
K3Fe(CN)6 and KCN
Hb + K3Fe(CN)6 → MetHb + KCN → CyanmetHb
 1. MEASUREMENT OF Hb LEVEL BY
CYANMETHB METHOD
Tube BLANK STANDARD Unknown
(mL) DUPLO DUPLO
(mL) (mL)

DRABKIN reagent (mL) 5.02 5 5

blood (mL) - - 0,02

STANDARD Hb (mL) - 0,02 -

Incubate at room 10 Minute 10 Minute 10 Minute

temperature
Read at 540 nm

Result : Hb level (g %)
MEASUREMENT OF HEMOGLOBIN LEVEL
Formula:

Au-Ab
Hb level= x C st (g%)
As-Ab
MEASUREMENT OF LIPID PEROXIDES
IN THE HEMOLYSATE
OBJECTIVES:
LEVELS SET PEROXIDES
LIPID IN BIOLOGICAL FLUIDS (hemolysate)
 PRINCIPLE
• Poli unsaturated fatty acids (PUFA) can
undergo lipid peroxidation which then
decomposes into malondialdehyde (MDA).
• MDA when reacted with TIOBARBITURAT acid
(TBA), will form a pink-colored compound that
absorbs light at wavelength: 532 nm.
• Total MDA that form can describe the process
of lipid peroxidation
 material BLANK U-1 U-2
(without t-BHP) (with t-BHP 10 mM)
duplo duplo

Aquadest 0.5 mL - -
Hemolysate without t-BHP - 0.5 mL -

Hemolysate with t-BHP 10 - - 0.5 mL

M
TCA solution 10 % 1 mL 1mL 1 mL
Shake it with vortex ,centrifuge, take the supernatant

TBA solution 0,67 % 1.5 mL 1.5 mL 1.5 mL

cover with marbles, input to the boiling bath, 10 minutes. cooled. Read the absorbance at
532 nm.

Result: count mda levels using formula

 formula
A
MDA level =
€
(€ =153.000 M-1 cm-1)
Sample preparation until centrifugation for
precipitate proteins using colorimetric tubes

• After centrifugation → separate the

supernatant by transferred to a regular test
tube
Recipes that are used (in the user guide)
multiplied by 2
tert-Butylhydroperoxide