determine the activity of G6Pase

CCN2231 – GENERAL BIOCHEMISTRY
Lecturer: Dr. IVAN CHUNG
GROUP PROJECT
LAB REPORT 1
Group B
Student Name: Yip Ho Yin (Student ID:12309985A)
Student Name: Wong Kin Hin (Student ID:12310084A)
Student Name: Ling Tung Yin (Student ID:12307848A)
Student Name: Cheuk Hiu Fung (Student ID:12309957A)
Student Name: Wong Hoi Tung (Student ID:12309501A)

Date of submission:

Date of Experiment: Introduction
Glucose-6-phosphase (G6Pase) is an enzyme presented in liver and used in the hydrolysis of glucose-6-phosphate (G6P). This catalyst the final step in glycogeneogeneis and glycogenolysis which is the key point of homeostatic regulating the blood glucose levels in our bodies.

In the experiment, we will determine the activity of G6Pase. Frist, we will extract the G6Pase from pig liver sample. Then, we will assay for the liver G6Pase by enzymatic digestion and phosphate determination. Finally, we will measure the amount of protein present in the G6Pase by the Bradford test and compare with the standard curve. We will also calculate the purity of the extracted enzyme according to the activity per unit protein.
Raw Data:
(B)

Diluted Pi standard 1mM (ml)
G6Pase sample (ml)
Water (ml)
Absorbance
(650nm)
(1) Reagent blank
-
-
7.6
0
(2) Std Pi (0.2 μmol)
0.2
-
7.4
0.122
(3) Std Pi (0.4 μmol)
0.4
-
7.2
0.243
(4) Std Pi (0.8 μmol)
0.8
-
6.8
0.563
(5) Std Pi (1.0 μmol)
1.0
-
6.6
0.617
(6) Std Pi (1.2 μmol)
1.2
-
6.4
0.883
(7) Liver “0” tube
-
0.5
7.1
0.055
(8) Liver “5” tube
-
0.5
7.1
0.063
(9) Liver “10” tube
-
0.5
7.1
0.072
(10) Liver “15” tube
-
0.5
7.1
0.090

(C)

Blank
Diluted standard
Diluted sample
Add amount (μl)
0
20
40
80
100
20
80
Add water (μl)
100
80
60
20
0
80
20
Absorbance (595nm)
0
0.152
0.294
0.367
0.745
0.035
0.168

Observation
In part 2(b), the tubes