University of Kirkuk Biochemistry /Practical

College of Dentistry Second Year / Group D

By:Abdullah Mohammed
Mohammed Rizgar
Yousif Mohammed Tahir
Noora Sirwan
Noora Ghazi
Israa Ali
Bnar waleed

2019/2020
GOD-PAP Test

Objective:
to determine the concentration of glucose in a sample of
serum .
Theory:
GOD-PAP Test: is A simple colorimetric method used to
determine the concentration of D-Glucose. Based on the
combined action of Glucose oxidase (GOD) and Peroxidase
(POD).
Glucose is oxidized by GOD to Gluconic acid and hydrogen
peroxide which in conjuction with POD , reacts with chloro-4-
phenol and PAP to form a red quinoneimine.

Glucose oxidase
Glucose + O2 Gluconic acid + H2O2
H2O2+4-amino-antipyrine+chloro-4-phenol
Red quinonimine + 4H2O

*The absorbance of the colored complex , is proportional to the

concentration of glucose in the specimen
We can measure the concentration of Unknown from this equation ,
since the absorbance changes with the concentration linearly.

( )
( ) ( )(( ))
(
GOD-PAP Test

Composition of the reagent:

1.R1 (working reagent)

1.phosphate buffer 150 mmol/L

2.glucose oxidase ≥ 20000 IU/L
3.peroxidase ≥ 1000 IU/L
4.4-amino-antipyrine 0.8 mmol / L
5.chloro-4-phenol 2 mmol / L

2. R2 : (standard)
glucose 100 mg/dl ( 5.55 mmol/l)
GOD-PAP Test

Apparatus: 3 test tubes , micropipettes , working

reagent , distilled water , spectrophotometer , sample of
serum .
GOD-PAP Test

Procedure
1.Take three test tubes and mark then Unkonwn(serum) ,
Standard , Blank .
2.Take 1 ml of working reagent in each tube.
3. Add 10 µl of distilled water into Blank .
4.Add 10 µl of Standard Solution into test tube marked as
Standard.
5.Add 10 µl of serum into the test tube marked as Unknown .
6.Mix and wait for 10-15 minutes , the colour will change to
red in unknown solution indicating the presence of chemical
reaction and the formation of red quinoneimine.
7.Use the blank solution to fix the reading of
Spectrophotometer at zero .using wavelength of 540 nm,
check the absorbance of Unknown and Standard .
GOD-PAP Test

Calculations :

Abs ( blank) = 0.00 mg/dl

Abs ( standard)=0.058
Abs( Unknown)=0.123
( )
( ) ( )(( ))
(

Concentration of glucose
GOD-PAP Test

Discussion and Conclusion :

In this experiment , we determined the concentration of
glucose in a sample of serum , by using colorimetric
(photometric) method . The experiment was based on the
fact that the absorbance of a solution , changes
proportionally to the concentration of the same solution at
At specific wavelength.
 The absorbance of the colored complex (red
quinoneimine) , is proportional to the concentration of
glucose in the specimen
Since we have standard solution of glucose (of known
concentration ) , and after measuring the absorbance of the
standard solution , then measuring the absorbance of
essay’s solution using spectrophotometer , we can easily
calculate the concentration of glucose in the sample of
serum from these information , which was about 212.06
mg/dl , however this was a temporary increase in blood
sugar , and does not indicate the presence of abnormal
condition like diabetes .