Sie sind auf Seite 1von 7

Experiment #5: Measuring the Binding Constant of

Ethidium Bromide to DNA

Abstract

In this lab, we measured the binding constant of ethidium bromide to DNA using

spectrophotometry. We did so by using a different amount of buffer solutions with different


concentrations of DNA, but we kept the concentration of ethidium bromide constant. Ethidium

bromide can bind to DNA by inserting itself between the base pairs in DNA. A

spectrophotometer is used in order to measure the absorbance of a solution. We expected to get

an s-shaped scatter plot of absorbance versus the log of the DNA concentration. The log of the

constant for our experimental values of absorbance was 5.12 the minimum absorbance was

0.0279, and the maximum absorbance was 0.0485.

Introduction

In this experiment, ethidium bromide will be used in order to bind with DNA. Ethidium

bromide can bind to DNA by inserting itself between the stacked bases in the double-stranded

DNA. It contains a tricyclic phenanthridine ring system, which is hydrophobic and resembles the

rings of the bases in DNA. Due to this hydrophobicity, ethidium bromide is able to bind to the

hydrophobic interior of the DNA molecule and thus is capable of forming close van der Waals

interactions with the base pairs. Ethidium bromide is considered an intercalating agent because

of this type of binding with molecules. These intercalating agents intercalate into the array of

stacked DNA bases. By intercalating the bases, these agents deform the DNA’s double helix,

which then causes an interference with transcription, replication, DNA repair, and

recombination.

Since the goal is for ethidium bromide to bind to DNA, herring testes DNA will be used.

Herring testes DNA (htDNA) is a natural DNA used in studies of DNA binding agents that

modulate DNA structure and function. Herring testes DNA will be used in different

concentrations over six orders of magnitude in this experiment. By doing so, it can be possible to

have almost completely unbound DNA and DNA, which is saturated with ethidium. The
concentration of ethidium that was bound can then be calculated by measuring the solutions

absorbance.

A spectrophotometer is used in order to measure the absorbance of a solution.

Spectrophotometry is a method used in order to measure how much a chemical substance

absorbs light by measuring the intensity of light as a beam of light passes through sample

solution in a spectrophotometer. The way this method works is that each compound either

absorbs or transmits light over a certain range of wavelength. This measurement of absorbance

can also be used on order to measure the amount of a known chemical substance. For these

reasons, a spectrophotometer will be used in order to calculate the amount of ethidium bromide

that was bound to the DNA. Each of the 19 samples that will be made will be placed in cuvettes

and placed in the spectrophotometer to measure their absorbance at 480nm.

Experimental Procedures

Obtain 19 cuvettes, 2mM and 2μM stock solutions of DNA, BPES buffer, and 1mM

stock solution of ethidium bromide. Prepare 19 different 1mL solutions containing the different

concentrations of DNA, BPES buffer, and 10.1μL of ethidium bromide in each cuvette. The

different concentrations and their corresponding amount of DNA and BPES buffer can be seen in

the table in the results section. Mix each solution and then measure their absorbance at 480nm.

Results

Concentration 2mM DNA (μL) BPES (μL) Absorbance

10-3 500 500 0.029


10-3.3 250 750 0.025

10-3.7 99.75 900.25 0.024

10-4 50 950 0.027

10-4.3 25 975 0.042

10-4.7 10 990 0.038

10-5 5 995 0.047

10-5.3 2.5 997.5 0.066

10-5.7 1 999 -0.002

Concentration 2𝛍M DNA (μL) BPES (μL) Absorbance

10-6 500 500 -0.001

10-6.3 250 750 0.041

10-6.7 99.75 900.25 0.051

10-7 50 950 0.047

10-7.3 25 975 0.049

10-7.7 10 990 0.046

10-8 5 995 0.049

10-8.3 2.5 997.5 0.063

10-8.7 1 999 0.092

10-9 0.5 999.5 0.062

Tables 1 and 2. These tables show the different concentrations and their corresponding amount
of DNA and BPES buffer that were used in the solutions. In the last column it shows their
absorbance that was measured in the spectrophotometer.
0.1

0.08
Absorbance

0.06

0.04
Series1

0.02

0
0 2 4 6 8 10

-0.02
-log (DNA BP)
Graph 1. The plot of absorbance versus the log of the DNA concentration.

Determination of Bound Ethidium Concentration


Absorbance: A=Eb(x)+Ef[E-x]
0.051=2497x+5600[0.000001-x]
0.051=2497x+0.56-5600x
-0.509=-3103x
x=0.000164M

Determination of Binding Constant


Binding Constant: K=[x]/[E-x][D-x]
K=[0.000164][0.000001-0.000164][0.0000002-0.000164]
K=[0.000164][-0.000064][-0.000162]
K=[0.000164][0.0000000104]
K=15800

Discussion
In this lab we wanted to measure the binding constant of ethidium bromide to DNA by

using spectrophotometry. The log of the constant that resulted from our data was 5.12, the

minimum absorbance was 0.0279, and the maximum absorbance was 0.0485. The scatter plot for

the absorbance versus the log of the DNA concentration that we expected to get using our data

was one that resembled an s-shape scatter. The results that we got do not exactly match that of an

s-shaped scatter plot but instead is somewhat linear. This difference in scatter plot can be due to

some sources of error which would probably have to do with either the spectrophotometer wasn’t

blanked correctly or it wasn’t accurately reading the absorbance at 480 nm. Also, human error

could have been at fault.

If BPE buffer is used instead of BPES, the binding constant would somewhat stay the

same. BPE buffer is BPES buffer without added NaCl and the salt concentration does not

interfere with the binding of BPE to DNA. Since ethidium bromide is a tightly packed molecule,

it can fit between the stacked bases of DNA with no problem.

The anticancer drug actinomycin D (act D) has an affinity for intercalation at the

sequence GC, which would lead to a lower binding concentration than those drugs that would be

non-specific and bind to everything. Since the act D searches for and binds to GC sequences, if

there is one GC base pair after another GC, act D can only bind to one of them. When

intercalating agents bind to a base pair they place a restraint in adjacent base pairs so no other

ligand can bind. This results in a higher concentration of free act D and free DNA receptor,

which means the constant would be significantly larger. The higher constant would lead to a

graph that looks like this:

References
Waring, M. J. (1965). Complex formation between ethidium bromide and nucleic acids. Journal

of molecular biology, 13(1), 269-282.

Reinhardt, C., & Krugh, T. (1978). A comparative study of ethidium bromide complexes with

dinucleotides and DNA: direct evidence for intercalation and nucleic acid sequence preferences.