Research Synopsis

Studies of Interaction and Oxidative Damage of Plant DNA with Some Metal (Fe,
Cr, Co, Mn) Citrate Complexes

Supervisor Dr. Md. Abdus Subhan

Professor (On leave)

Department of Chemistry

Shahjalal University of Science and Technology Sylhet,

Bangladesh

Submitted by Md. Saifur Rahaman

Department of chemistry

Reg. No. 2008121026

SUST, Sylhet, Bangladesh

Introduction

Transition metal complexes that are suitable for binding and cleaving double strand DNA [1] are
of considerable current interests due to their various applications in nucleic acid chemistry like
foot-printing and sequence-specific binding agents, for modeling the restriction enzymes in
genomic research, and as structural probes for therapeutic applications in cancer treatment. Metal
complexes can also be used to study the oxidative damage of DNA and cellular membrane which
cause cancer and aging [2-4] in higher organisms which produces interests in designing drugs
which have mainly antioxidant properties. The mechanism of oxidative damage of DNA is given
in the following figure (Fig -1). Oxidative stress is caused by an imbalance between the
production of reactive oxygen and a biological system's ability to readily detoxify the reactive
intermediates or easily repair the resulting damage. All forms of life maintain a reducing
environment within their cells. This reducing environment is preserved by enzymes that maintain
the reduced state through a constant input of metabolic energy. Disturbances in this normal redox
state can cause toxic effects through the production of peroxides and free radicals that damage all
components of the cell, including proteins, lipids, and DNA. DNA oxidative damage by reactive
oxygen species causes extensive strand breaks in DNA. [5-7] Oxidative stress is involved in many
diseases, such as atherosclerosis, Parkinson's disease, heart failure, myocardial infarction,
Alzheimer's disease, fragile X syndrome and chronic fatigue syndrome, but short-term oxidative
stress may also be important in prevention of aging by induction of a process named
mitohormesis. The main view of our present study is to study the interaction as well as the
oxidative damage of DNA by metal citrate complexes. Citric acid, a naturally occurring
compound, is found in solids and plant exudates and is produced microorganisms, especially
fungi. Citric acid has been widely known for its abundance in physiological fluids and its multi
potent chemical versatility toward biologically relevant metal ions. [8] It actively participates in
the Krebs cycle and is an essential component in a number of processes occurring in biological
systems as in the carbohydrate metabolism in the citric acid cycle as a substrate, in mutant forms
of the nitrogenase enzyme as an organic ligand, in aconitase enzymes. And its importance in the
above biological systems remarks its significance as an important component in a number of
metalloenzyme sites. It presents a chemically attractive ligand target to a number of trace metal
ions circulating in biological fluids, including those in humans. Complexation to such metal ions
begets solubility, potential bioavailability, and likely absorption to biological sites bybiological
tissues in need of those essential metals. Till the present time many metal citrate complexes have
been synthesized and studied but the binding properties of the metal citrate complexes has not
been studied thoroughly yet. So we intend to study the interaction and oxidative damage of plant
DNA with metal citrate complexes with a view to synthesizing drugs of medicinal interests.
 Fig-1- Mechanism of oxidative damage by reactive oxygen species

Objectives

This project has aims

 To investigate the binding properties of plant DNA with metal (Fe, Cr, Co and Mn)
citrate complexes Using UV-Visible spectroscopy, viscosity measurement and DNA
electrophoresis.
 To investigate the damage of DNA by metal citrate complexes.
 To investigate the cleavage properties of plant DNA by metal citrate complexes.
 To investigate the probability in designing citric acid and metal citrate complex related
active phermaceutical ingredient.

Methodology

1. Synthesis of metal complexes

All the metal complexes will be synthesized by the reaction of corresponding metal (Fe, Cr, Co,
Mn) salts with citric acid with a 1:2 molar ratio in aqueous solution at pH ranges from 5.5~ 8 [9-12]
All the reaction can be represented with a single representative reaction scheme

M n+ + citric acid

Charecterization of the metal complexes

The synthesized complexes will be charecterized by UV-Visible, IR, Conductance measurement

and other physical perameter.

2. Study of DNA-metal citrate complex interaction

a) UV-Visible spectrophotometric study: This study will reveal the binding ability of the
metal-citrate complex with DNA. This study will also disclose the difference between the
binding ability of different metal citrate complexes with DNA. In this context we have to make
use of the following equation-
[DNA]/(Ԑa-Ԑf) = [DNA]/(Ԑb-Ԑf) + 1/Kb(Ԑa-Ԑf)
Where, [DNA] is the concentration of DNA, Ԑa is the apparent extinction coefficient which can
be obtained by calculating Aobs/[complex], Ԑf is the extinction coefficient of the complex in its
free form, kb is the binding constant of the metal complex to DNA and Ԑ b is the extinction
coefficient of the complex in the fully bound form.
Each set of data, when fitted to the above equation, gave straight line with a slope of 1/ (Ԑ a-Ԑf)
and a y-intercept of 1/Kb (Ԑa-Ԑf). Thus from the graph the binding constant Kb can be determined.

b) Viscometric study: This study will reveal the binding modes of the metal complex to DNA
that is whether the binding of the metal citrate complex to DNA is intercalative or electrostatic or
both. In this context we will make use of the following equation-
η = πPr4t/8vl and
Where η is the viscosity of a liquid, P is the driving pressure of the liquid that makes the liquid to
flow, v is the velocity, r is the radius, t is the time and l is the length. Thus getting the viscosity
of the blank solution we have to use the following equation to get the viscosity of our sample
solutions.
η1/ η2= ρ1t2/ρ2t2
Thus by using the above equations and comparing the viscosities of blank solution, DNA
solution, metal citrate complex solution and metal complex –DNA solutions we will be able to
make an inference about the binding modes of the metal citrate complex with DNA.

c) DNA electrophoresis: This study reveals the important property of DNA- metal complex
binding and that is cleavage of DNA by the interaction of metal citrate complex with plant DNA.

3) DNA oxidative damage study

We intend to study the oxidative damage of plant DNA caused by the metal citrate complexes
themselves and also in presence of H2O2. Metal citrate complexes can damage oxidatively by
producing reactive oxygen species in presence or absence of H2O2. The mechanism of reactive
oxygen species production is given below
Fe2+ + H2O2 → Fe3+ + .OH + OH-
Fe3+ + H2O2 → Fe2+ + .OOH + H+
We will study the oxidative damage of DNA by means of DNA electrophoresis

Socio economic Contribution

Citric is widely used in foods in beverages as an acidulation agent and preservative. So this
project will help us improving our research skills in chemistry as well as the biological section of
chemsitry. This project will help making new drugs containing citric acid of pharmaceutical
interests as we are investigating the probability in designing metal citrate complexes related
drugs. Therefore, because of the wide importance and use of citric acid the skill and knowledge
generated by this research works can be used in the development of the pharmaceuticals industry
in Bangladesh.
Time Frame

The work will be done within one year as the research plan chart:

Month 1st 2n 3r 4th 5th 6th 7th 8th 9th 10th 11th 12th
d d
Synthesis and × × ×
charecterization of metal
complexes
DNA Extraction from ×
plant
Study of the DNA × × × × ×
interection and damage
by mentioned methods.
Data analysis and thesis × ×
paper writing
Thesis paper presentation ×

Conclusion

The aim of this work is to investigate the interaction as well as the oxidative damage properties
of metal citrate complexes with plant DNA to find out the probability of designing new drugs
mainly of antioxidant properties. Binding characteristic will be investigated by UV-Visible
spectroscopy, viscometry and electrophoresis.

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