ESTIMATION OF ANTIOXIDANT ENZYME (CATALASE):

A BIOMARKER FOR TYPE 2 DIABETES

MAJOR PROJECT REPORT
SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENT FOR THE DEGREE

BACHELOR OF TECHNOLOGY
IN
BIOTECHNOLOGY
BY
SHAYISTA NAAZ
09J21A2316

DEPARTMENT OF BIOTECHNOLOGY
NATIONAL BOARD OF ACCREDITATION (NBA)

JOGINPALLY B.R ENGINEERING COLLEGE

(Affiliated to Jawaharlal Nehru technological university, Hyd, A.P)
Yenkapally, Moinabad Mandal Ranga Reddy district-500-075

JOGINPALLY B.R ENGINEERING COLLEGE

(Affiliated to Jawaharlal Nehru technological university, Hyd, A.P)
Yenkapally, Moinabad Mandal, Ranga Reddy district-500075

DEPARTMENT OF BIOTECHNOLOGY
CERTIFICATE
This is to certify entitled ESTIMATION OF ANTIOXIDANT ENZYME
(CATALASE): A BIOMARKER FOR TYPE 2 DIABETES is a bonafide work
carried at, JNIAS HYDERABAD, by Ms. SHAYISTA NAAZ, bearing the registered
number 09J21A2316, in partial fulfillment of the requirements for the degree
BACHELOR OF TECHNOLOGY in BIOTECHNOLOGY from Joginpally B.R
Engineering College, Yenkapally, affiliated to Jawaharlal Nehru Technological
University, Hyderabad, during the academic year 2012-2013.

Internal Guide

Head of the Department

ACKNOWLEDGMENT
Every successful task needs some support. This project was undertaken with the active
support of staff of Jawaharlal Nehru Institute of Advanced Studies Hyderabad (A.P).
I express my sincere gratitude to my project guide

Dean of School of Life

Science, Dr. KAISER JAMIL, for sparing her valuable time in giving valuable
information and suggestion for the successful completion of my project.
I would also like to express my deep gratitude to my research scholar Mr.
MD.ASSIMUDDIN for his inspiring guidance and encouragement.
I convey my gratitude to my internal guide Ch.N.VANI, Assistant professor, Biotech
Department, for allowing me to do project and enabling me to complete the project
successfully. I express my profound gratitude for her valuable guidance and support.
My sincere thanks to Ms. Dr. ZEHRA SIDDIQUI, Associate Professor, Biotechnology
Department, Project coordinator, for her valuable suggestions and advices throughout my
project work.
I hereby thank one and all who extended help hand in the accomplishment of the project.

SHAYISTA NAAZ

DECLARATION

I hereby declare that the project work entitled ESTIMATION OF ANTIOXIDANT

ENZYME (CATALASE): A BIOMARKER FOR TYPE 2 DIABETES is original
and has carried out by me under the supervision of Dr. KAISER JAMIL, Dean Of
School of Life Science, Jawaharlal Nehru Institute Of Advanced Studies,
Hyderabad, during the academic year 2012-2013. This work presented here has been
submitted in partial fulfillment for the award of Bachelor of Technology Degree in
Biotechnology from Jawaharlal Technological University, Hyderabad, Andhra Pradesh.

SHAYISTA NAAZ
(09J21A2316)

INDEX
Chapter No.
1

Content

Page no

Introduction
1
2
3
4
5
6

1-12

Diabetes
1
Causes of Diabetes
Risk Factors for Type 2 Diabetes
Reactive Oxygen Species (ROS)
Antioxidants And Antioxidant-Related Enzymes
Catalase
8
1.7

2
3
5
6

Catalase and Diabetes

11

1.8

Aim and Objective

12

Review of literature

13-16

Materials and Method

17-30
3.1 Biochemical Analysis of Catalase
17

3.1.1 Estimation of Protein in Total Serum Sample

17
3.1.2 Estimation of Catalase
19

3.2 Molecular Analysis of Catalase

20
3.2.1 Isolation of Genomic DNA from Human
Blood Sample
20
3.2.2 Agarose gel electrophoresis
23

3.2.3 Spectrophotometer
25
3.2.4 Polymerase Chain Reaction
26

Result and Discussion

4.1 Biochemical analysis

31-40

31

4.1.1 Protein Standard graph

31
4.1.2 Estimation of Total protein serum analysis
32
4.1.3 Relationship between Catalase activity and
Glucose level

33

4.2 Molecular analysis

35
4.2.1 Isolation of DNA from Normal and Diabetic
Blood Samples

35

4.2.2 Quantification of DNA by Spectrophotometer

36
4.2.3 Identification of Catalase Gene by
PCR Amplification
38

Conclusion

41

Reference

42-45

List of Figures:
Figure
Number

Figure Name

Page No

Schematic representation of onset of

type 1 diabetes
Schematic representation of onset of
type 1 diabetes
Schematic representation of the
major players of the cellular antioxidant network.
Schematic representation of
catalase gene located on
chromosome 11: base pairs
34,460,471 to 34,493,606.
Catalase gene Mapping

10

Figure 7
Figure 8

Schematic representation of 3D
catalase structure
Agarose gel electrophoresis
Gel documentation system

Figure 9

Spectrophotometer

25

Figure 10

Polymerase Chain Reaction

28

Figure 11

The exponential amplification of the

29

Figure12

gene in PCR.
Standard graph

31

Figure 13

Total Protein in Serum

32

Figure 14

Catalase activity in normal and

34

Figure 1
Figure 2
Figure 3
Figure 4

Figure 5
Figure 6

2
7
9

24
24

diabetic
Figure 15
Figure 16
Figure 17
Figure18

Isolated genomic DNA from normal

blood samples
Isolated genomic DNA from diabetic blood
samples
PCR product of normal individual
(a): PCR product of diabetic
individual
(b): PCR product of diabetic
individual

35
36
39
40

List of Tables:

Table
Number

Table Name

Page
No

Table 1

Reactive oxygen species sources and

their products

Table 2

Setup of different dilution for

18

standard graph
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8

21
RBC Lysis Buffer/ TKM 1
Cell Lysis Buffer/ TKM2

21

Setup of different dilution of

reagents and protein for standard
graph
Spectrophotometer value of Normal
serum sample of Catalase activity

33

Spectrophotometer value of Diabetic

serum sample of Catalase activity
showing purity and concentration of
DNA of 8 normal individuals by
quantifying with UV
spectrophotometer

34

33

37

Table 9

Showing DNA Purity and

concentration of 7 diabetic
individuals by quantifying with UV
spectrophotometer

ABBREVATIONS
1. DNA : Deoxyribo nucleic Acid
2. EDTA : Ethylene Diamine Tetra-acetic Acid
3. TAE : Tris- Acetate EDTA
4. PCR : Polymerase Chain Reaction
5. AGE : Agarose Gel Electrophoresis
6. SDS : Sodium Dodecyl Sulphate
7. DNTPs : Di Deoxy Nucleotide Tri Phosphate
8. BSA : Bovine Serum Albumin
9. O.D
: Optical Density
10. KB
: Kilo Base Pairs
11. ROS : Reactive Oxygen Species
12. OS
: Oxidative Stress
13. bp
: base pair
14. CAT : Catalase
15. l
: micro litter
16. mg
: milligram
17. sec
: second
18. Conc. : Concentration
19.
NaOH : Sodium hydroxide

37

ABSTRACT
Diabetes is a prevalent systemic disease affecting a significant
proportion of the population worldwide. It has been suggested that
enhanced production of reactive oxygen species (ROS) and oxidative
stress is central event to the development of diabetic complications.
Recent studies have shown that high reactivity of ROS determines
chemical changes in virtually all cellular components, leading to lipid
peroxidation. Production of ROS and disturbed capacity of antioxidant
defense in diabetic subjects have been reported. Use of antioxidants
reduces

oxidative

stress

and

alleviates

diabetic

complications.

Catalase, an antioxidant, catalyzes the reduction of hydroperoxides,

thereby protecting mammalian cells against oxidative damage. In
addition, Catalase is active in neutralizing reactive oxygen species and
so removes cellular superoxide and peroxides before they react with
metal catalysts to form more reactive species. In the present study we
estimated the effect of antioxidant (catalase) activity in type 2
diabetes and found that increase in glucose conc. shows decrease in
catalase activity. Therefore catalase gene may be used as potential
biomarker for Type 2 Diabetes.
Keywords: Antioxidant (Catalase), reactive oxygen species (ROS),
type 2 diabetes.