1

Spectroscopic Analysis of the Interaction

between Dihydropteridine Reductase

(DHPR) its Cofactor and Folic Acid (FA)

Tharenie Sivarajah Thesis Paper Mentor: Dr.Desamero Major: Chemistry May 4th 2007

2

TABLE OF CONTENTS

Chapters Page

Preface ……………….. 3

Introduction ……………….. 4

Principles behind the UV-VIS, IR and fluorescence ……………….. 6

Characteristics and properties of the protein and inhibitors studied ……………….. 14

Results from UV-VIS and fluorescent work ……………….. 20

IR studies & Calculated simulations for folic acid ……………….. 39

Conclusions ……………….. 47

References ……………….. 48

3

Chapter 1

Spectroscopic Analysis of the Interaction between

Dihydropteridine Reductase (DHPR) its Cofactor and Folic

Acid (FA)

Preface

My work centered on exploring the interaction between the enzyme

dihydropteridine reductase (DHPR), cofactor NADH, and ligands, which in my case was

folic acid (FA). DHPR is an enzyme which is important in a biochemical pathway that

recycles a substance called tetrahydrobiopterin. Tetrahydrobiopterin is important in the

conversion of precursors of neurotransmitter like serotin. Deficiency in DHPR leads to

phenylketonuria disease (PKU).

Enzyme and inhibitor interactions were observed both in the presence and the

absent of the cofactor. The third chapter of my thesis will include a discussion about the

principles behind the UV-VIS, IR and fluorescence spectroscopy. In the fourth chapter,

I’ll talk about the characteristics and properties of the protein and inhibitors studied.

Chapter 5 will focus on the result we obtained from our UV-VIS and fluorescence work.

Chapter 6 will be devoted to the IR studies. In both chapters 6 and 7, critical analysis of

the data and the conclusion drawn from the results will be presented. The last chapter

will include the calculated simulations for folic acid. This will support and explain the

results presented in chapters 6 and 7.

4

Chapter 2

Introduction

The overall goal of our research is to understand the interplay between protein

structure, inhibitor, and the cofactor. The enzyme Dihydropteridine reductase (DHPR)

was studied in a model system. DHPR is needed for the normal cycling of

tetrahydrobiopterin, which is a very important cofactor in the brain. If we don't have

tetrahydrobiopterin then we won't have neurotransmitters such as serotonin. Serotonin is

essential for the brain functioning. In humans, DHPR deficiency causes a neurological

disorder known as phenylketonuria (PKU). PKU is a genetic disorder that is

characterized by an inability of the body to utilize the essential amino acid,

phenylalanine. Amino acids are the building blocks for body proteins. Essential amino

acids can only be obtained from the food we eat as our body does not normally produce

them. In classic PKU, the enzyme phenylalanine hydroxylase is completely deficient,

which breaks down phenylalanine. This enzyme normally converts phenylalanine to

another amino acid, tyrosine. Without this enzyme, phenylalanine and its breakdown

chemicals from other enzyme routes, accumulate in the blood and body tissues and result

in PKU.

Fluorescence spectroscopy plays an important role in investigating the interaction

between proteins (enzymes), and small molecules, such as ligands and cofactors. We

compared the DHPR with inhibitor and with both inhibitor and cofactor with the help of

fluorescence. Data showed that a change in fluorescence intensity and fluorescence

5

peaks results when the cofactors and/or inhibitors were complex with DHPR. Infrared

spectroscopy was used to identify the IR bands in the ligand folic acid. Calculated

simulations for folic acid and its protonated form were obtained from the Gaussian03

program in order to compare the experimental data of folic acid and see which bonds are

interacting when the ligand is within the protein.

6

Chapter 3

Theory behind spectroscopy

Spectroscopy is a type of analysis done by shining the light on the molecule

(sample) to learn more about it and to see what is inside. In spectroscopy, chemists

measure the absorbance, amount of light passes and absorbed by the sample. By

knowing what wavelengths of lights are absorbed by the sample, we can learn more about

that sample. On the other hand, if we shine the light at the wrong wavelength of light to

study about the specific characteristic of a molecule then we will never learn anything

about that molecule because different characteristics of molecules absorb energies at

different wavelengths of lights. That’s why there is a need for different forms of

spectroscopy such as, ultraviolet spectroscopy (UV), fluorescence spectroscopy, and

infrared spectroscopy (IR). These different forms of spectroscopy investigate specific

characteristics of a sample. For example, IR spectroscopy is used to examine how the

molecules in a sample vibrate, while ultraviolet spectroscopy (UV) is used to investigate

how certain chemical bonds in a molecule are arranged.

UV-Vis spectroscopy

UV range is from 200 to 400 nm and visible (VIS) range is 400 to 800 nm. UV

spectroscopy is useful to investigate electronic structure of unsaturated molecules and to

determine the amount of conjugation in a molecule. In our research, we use it to see the

absorption peak for the compound so that we can excite the compound at the absorption

peak in fluorescence. Absorption of ultraviolet or visible light by a molecule depends on

7

electron transitions between molecular orbital energy levels. Absorption spectroscopy is

mostly based on electronic transitions of non-bonding (n) or bonding (π) to the anti

bonding (π*) excited state because these transitions fall in the region of 200-700nm. For

example, C=C is a π to π* transition and C=O is a n to π* transition. When an electron

goes from a higher to a lower energy state, a photon of definite wavelength and frequency

is emitted. However, a photon is absorbed when electromagnetic radiation in the UV-Vis

range passes through a molecule containing multiple bonds. As a result, in folic acid,

photon is absorbed due to its double and carbonyl bond. Therefore, amount of energy

absorbed depend on the compound structure. Molar absorptivity (ε) measures how

strongly the species absorb light at a given wavelength. My ligand folic acid has a molar

absorptivity of 7.95 x 103M-1cm-1 . Every molecule has a characteristic electronic

spectrum depending on its characteristic ΔEs. There is a general rule that describes the

effect of double bonds conjugation on the energy absorbed by the π system which is the

greater the number of conjugated multiple bonds in a compound, the longer the

wavelength of the light that the compound will absorb. Folic acid has seven conjugated

bonds from the rings and thus has an absorption peak at 363 nm. Schematic diagram of

UV-VIS spectrometer is shown in figure one.

8

Figure #1

1

The function of the UV instrument is relatively straightforward. It has the source,

the sample and the detector. A beam of light from a UV light source is separated into its

component wavelengths by a diffraction grating or prism. Each monochromatic (single

wavelength) beam is split into two equal intensity beams. One beam is the sample beam

where we place the cuvette with our sample and the other beam is the reference beam

where we place the cuvette with our solvent only. Detectors then measure the intensities

of these light beams.

Fluorescence spectroscopy

Fluorescence spectroscopy is a type of electromagnetic spectroscopy used for

analyzing fluorescent spectra. It is primarily concerned with electronic states and

vibrational states. In fluorescence spectroscopy, the electron is first excited by absorbing

a photon of light from its ground electronic state to one of the various vibrational states in

the excited electronic state. Collisions with other molecules cause the excited molecule

to lose vibrational energy until it reaches the lowest vibrational state of the excited

1 Spectroscopy – UV/Vis. http://www.sci.sdsu.edu/TFrey/Bio750/UV-VisSpectroscopy.html

9

electronic state. The molecule then drops down to one of the various vibrational levels of

the ground electronic state again, emitting a photon in the process. As molecules drop

down into any of the vibrational levels of this ground state, the photons will have

different energies, and frequencies. Therefore, by analyzing the different frequencies of

light emitted in fluorescent spectroscopy, the structure of these different vibrational levels

can be determined.

When electrons go from the excited state to the ground state, there is a loss of

vibrational energy. As a result, the emission spectrum is shifted to longer wavelengths

than the excitation spectrum. Emission and excitation spectra are obtained from

fluorescence spectroscopy. Emission spectrum is wavelengths emitted by fluorophore

after it has been excited by a photon of light. Fluorophore is the specific region of a

molecule, which is capable of exhibiting fluorescence. The fluorescence emission

spectrum is plotted as relative intensity as a function of wavelength and it is positioned in

longer wavelengths than the excitation spectrum. Excitation spectra are generated by

scanning through the absorption spectrum of a molecule while monitoring the emission at

a single peak. In a similar manner to the absorption spectrum, the excitation is broadened

due to vibrational and rotational relaxation of excited molecules. Absorption and

excitation spectra are distinct but they often overlap. Schematic diagram of a fluorometer

is shown in figure two.

10

2

Fluorescence instrument has the source, the sample and the detector as in

UV/VIS. First, the light would pass through an excitation filter and then it will be

absorbed by the sample. After the excitation, fluorescence will be emitted. Then, light

would pass through the emission filter and the amount of light pass through can be

measured by the detector.

2 Fluorometers. http://www.iugaza.edu/users/abdellatif/Instrumental%20Analysis/Lectures/Lectures%2021-/L30.pdf

11

Figure #3

Jablonski diagram3

Jablonski diagram is shown in figure three and it basically shows the electronic

states and the transitions between them in a molecule. The states are arranged vertically

by energy and grouped horizontally by the spin multiplicity. Straight arrows indicate the

radiative transitions while the squiggly arrows indicate the nonradiative transitions.

When the fluorophore absorbs energy, it is excited to a higher vibrational energy level in

the first singlet excited state (S1) before it goes to the lowest energy level and this process

is known as absorption. Internal conversion takes place when there is a transition

between energy states of the same spin state (S2 • S1). On the other hand, intersystem

crossing transition takes place between different spin states (S1 • T2 ). Process of photon

emission between states of the same spin state is known as fluorescence (S1 • S0 ) and

the photo emission between different spin state is knows as phosphorescence (T1 • S0 ).

3 Jablonski Diagram. Research\Jablonski Diagram.htm.

12

Infrared spectroscopy

Infrared spectroscopy deals with the infrared region of the electromagnetic

spectrum. It is used to identify a compound and to investigate the composition of a

sample. The infrared portion of the electromagnetic spectrum is divided into three

regions which are near, mid and far infrared. The far-infrared lies in the region of

400-10 cm-1, mid-infrared is from 4000-400 cm-1 and the near-infrared is from 14000-

4000 cm-1 . Infrared spectroscopy works because chemical bonds have specific

frequencies at which they vibrate corresponding to energy levels. Vibrational frequencies

depend upon the strengths and geometry of the chemical bonds. The energy of a

vibration is measured by its amplitude so the higher the vibrational energy, the larger the

amplitude of the motion.

Only the bond that exhibits the change in dipole moment when it vibrates would

be seen in IR. For instance, asymmetric stretches will not be seen in IR since there will

be no change in dipole moment. In order to measure a sample, a beam of infrared light is

passed through the sample, and the amount of energy absorbed at each wavelength is

recorded. This can be done by using a Fourier transform instrument to measure all

wavelengths at once. From this, a transmittance or absorbance spectrum would be

plotted, which shows at which wavelengths the sample absorbs the IR, and allows an

interpretation of which bonds are present. Schematic illustration of FT-IR is presented in

figure four.

13

Figure #4

4

A Fourier Transform Infrared (FTIR) spectrometer obtains infrared spectra by

collecting sample signal with an interferometer. It measures the frequencies of infrared at

the same time. An FTIR spectrometer obtains the interferogram, carries out the Fourier

Transform function, and displays the spectrum.

4 Infrared Spectroscopy. http://sis.bris.ac.uk/~sd9319/spec/IR.htm

14

Chapter 4

Characteristics and properties of the protein and inhibitors studied

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of

tetrahydrobiopterin, which is the cofactor of the aromatic amino acid hydroxylases.

Quite number of studies has been done on DHPR. The studies show that monomeric

DHPR has a Km value of 7.8 x 10-5M and the dimeric DHPR has a km value of

1.1 x 10-5 M. Studies also show the molecular weights of the monomeric and the dimeric

enzymes are 26,000 and 52,000 respectively. 5

We are currently working on the protein extraction from rat liver. The intention

of this project was to extract the protein dyhydropteridine reductase (DHPR) so that it can

be used in our research to govern the interaction within protein, ligand, and cofactor.

Centrifugation, extraction, and dialysis methods were used mainly throughout this

project. After dialysis, UV peak at 280 nm showed the indication of protein peak.

However, this project is not completed yet.

5 Higher Education Commission. http://www.hec.gov.pk/htmls/thesis/thesis_detail.asp?op=72

15

Figure #5

Dihydropteridine Reductase (DHPR)

O OH

HN

N N H

H N

OH H2N

N N

OH

O OH

5,6,7,8-tetrahydrobiopterin

NADH, H+

NAD+

H2O

O2

dihydropteridinereductase

tyrosine

L-dopa

tryptophan

phenylalanine

tyrosine

5-hydroxytryptophan serotonin cathecholamines (dopamine, epineprine,norepineprine)

1. phenylalanine hydroxylase

3. tryptophan hydroxylase 2. tyrosine hydroxylase

H2N N N H

quinonoid-dihydrobiopterin

Dihydropteridine Reductase (DHPR) is shown in figure five and this cycle is

showing us that why the dihydropteridine reductase (DHPR) enzyme is important. As

you can see, DHPR recycles Tetrahydrobiopterin which is the important cofactor in the

brain. Tetrahydrobiopterin is essential in the conversion of aromatic amino acids to

precursors of neurotransmitters like serotin, which is important for the brain function. It

is also essential in the conversion of phenylalanine to tyrosine by the enzyme

16

phenylalanine hydroxylase. Therefore, defect in the DHPR enzyme or the lack of

phenylalanine hydroxylase causes phenylketonuria (PKU).

Figure #6

Crystal Structure of DHPR

Apo form of x-ray crystal structure of the DHPR, which is superimposed to that

of the NADH bound-form, is shown above. The blue ribbon represents the enzyme

DHPR by itself while the one in green are the domains that shift upon NADH

binding. This shift upon NADH binding triggered us to study the interaction between

the protein structure DHPR, cofactor NADH, and the ligand folic acid. However, the

17

Crystal structure of the substrate bound enzyme has not been solved. Therefore, it

would be our long-term goal to find the crystal structure of the ternary system

involving enzyme/cofactor and the substrate.

Figure #7

Ligand Folic Acid (FA)

O COOH NH2 C

N COOH N H N N H

NH2 N N (a)

(b)

18

Structure of folic (a) acid and its 3D form (b) is shown on figure seven. As you

can see, it has carboxylic and amine functional groups as well with two fused and one

separate benzene rings. Folic acid resembles similar structure as the substrate

dihydrobiopterin and thus would be important to study folic acid. Folic acid is involved

in the formation of new cells and therefore essential for the normal growth and

development of the fetus. Folic acid is active as a dietary component, but must be

metabolized to the reduced dihydrofolate and tetrahydrofolate forms for biological

activity. A deficiency of folic acid impairs DNA synthesis and cell division.

Figure #8

Structure of the cofactor NADH

C NH2 O-

O O P O

N+

O O

OH OH

O P O

O

HO

N

N

N

N

NH2

HO

19

Beta-nicotinamide adenine dinucleotide (NADH) is shown in figure eighteen.

NADH is an important cofactor that helps enzymes in the work they do throughout the

body. Each of the body's cells contains an energy powerhouse called the mitochondria.

A series of reactions called the energy electron transfer chain that takes place in the

mitochondria to produce cellular energy in the form of ATP. NADH plays a key role in

this process. It also participates in the production of L-dopa, which the body turns into

the important neurotransmitter dopamine.

20

Chapter 5

Results from UV-VIS and Fluorescence

One of our objectives is to see whether our ligand folic acid is stable at various

temperatures with respect to change in time because if our ligand is not stable then it

would be hard for us to govern the protein-ligand interaction. Therefore, we ran the folic

acid with a pH of 7.40 at 4, 25 and 440C for two hours and observed the folic acid to be

stable (Figure 9a, b and c).

Figure #9a

50µM Folic Acid Emission Ex 280 At 4C

10

9

8 0 min

7 30 min

6 60 min

90 min 5

120 min 4

3

2

1

0 320 370 420 470 520

Wavelength (nm)

Inte

nsi

ty

21

The fluorescence spectrum of 50µM Folic Acid that was excited at 280 nm and

ran at 4 0C displays two fixed peaks at 360 nm and 450 nm regardless of increase in time

(Figure #9a). It demonstrates the stability of ligand folic acid at 4 0C since there are no

changes in the intensity levels or in the peak positions.

Figure #9b

50µM Folic Acid Emission Ex 280 At 25C

10

9

8 0 min

7 30 min

60 min 6

90 min 5

120 min

4

3

2

1

0 320 370 420 470 520

Wavelength (nm)

Inte

ns

ity

22

Figure nine (b) shows the fluorescence spectrum of 50µM Folic Acid

which was excited at 280 nm and ran at 25 0C. It displays two fixed peaks at 360 nm and

450 nm regardless of increase in time and thus the folic acid is stable at 25 0C because

there are no changes in the intensity levels or in the peak positions.

Figure #9c

50µM Folic Acid Emission Ex 280 At 44C

10

9

8 0 min

30 min 7

60 min 6

90 min 5

120 min

4

3

2

1

0 320 370 420 470 520

Wavelength (nm)

Inte

ns

ity

UV for 3µM folic acid

360 nm 280 nm 23

Figure nine (c) illustrates the fluorescence spectrum of 50µM Folic Acid which

was excited at 280 nm and ran at 44 0C. It also displays two fixed peaks at 360 nm and

450 nm with no change in the intensity levels regardless of change in time. Therefore,

ligand folic acid is also stable at 44 0C.

UV for folic acid

Absorbance of folic acid (3µM), binary (3µM folic acid + 0.7µM DHPR), and

ternary (3µM folic acid + 0.7µM DHPR + 3µM NADH) system with the pH of 7.40 were

ran in UV and peaks were observed at 280 and 360 nm. These UV spectra were

presented in figure 10 a, b and c respectively.

Figure #10a

Abs

orba

nce

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 200 250 300 350 400 450 500

Wavelength Figure ten (a) shows the peaks of 280nm and 360nm with the absorbance

of 0.87 and 0.30 respectively.

UV FOR BINARY (3µM FOLIC+0.7uM DH

24

Figure #10 b

PR) 280 nm

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Ab

so

rban

ce

360 nm

240 290 340 390 440 490 540 590 640 690

Wavelength (nm)

Peaks were revealed at 280 nm and 360 nm with the absorbance of

0.33 and 0.14 respectively (Figure #10 b). The absorbance of folic acid is lower when it’s

in binary than when it is by it self.

UV For Ternary (3µM Folic, 0.7µM DHPR, 3µM NADH)System

25

Figure #10 c

280 nm

Ab

so

rba

nc

e

3

2.5

2

1.5

1

0.5

0

360 nm

200 250 300 350 400 450 500 550 600 650

Wavelength

Peaks were observed at 280 nm and 360 nm with the absorbance of 0.25

and 0.85 respectively (Figure #10 c). The absorbance of folic acid is higher when it’s in

ternary than when it is by it self and in binary.

700

26

445 nm

Inte

ns

ity

Folic acid in fluorescence

Folic acid (3µM), binary (3µM folic acid + 0.7µM DHPR), and ternary (3µM

folic acid + 0.7µM DHPR + 3µM NADH) system with the pH of 7.40 were excited at

280nm, 340nm and 360nm under the temperature dependent in fluorescence. These

spectra were presented in 11 a, b, c, d, e, f, g, h, and i respectively. Then, Folic acid

(3µM), binary (3µM folic acid + 0.7µM DHPR), and ternary (3µM folic acid + 0.7µM

DHPR + 3µM NADH) system with the pH of 7.40 were emitted at 345 nm and 445nm

and exhibited the peak at 280nm, 345 nm and 360 nm respectively. These excitation

spectra are shown in figure 12a, b, c, d, e, and f. In all of the spectra, observed the

decrease in intensity when the temperature was raised.

Figure #11a

3µM Folic Acid Temperature dependent Emission Ex 280 nm

4C 4.50 14C

24C 3.50 34C

2.50 44C

1.50

0.50 380 400 420 440 460 480 500 520 540

Wavelength (nm)

27

445 nm

Figure 11 (a) illustrates the temperature dependent emission at 280nm for

3µM folic acid. Peak was observed at 445 nm with the intensity of 2.95.

Decrease in intensity was observed with the increase in temperature.

Figure #11b

3µM Folic Acid Temperature dependent Emission Ex 340 nm

3 4C

14C

ty 2 24C

ns

i

34C

Inte

1 44C

0 385 435 485 535 585

Wavelength (nm)

Folic acid (3µM) was excited at 340 nm under various temperatures (4, 14, 24, 34

and 44 0C) and the peak was observed at 445 nm with the intensity of 1.95 (Figure #11b).

Intensity level was decreased when the temperature was increased.

28

445 nm

2 4C

y 14C

sit 24C

en 1.5 34C

Int

44C

1

Figure #11c

3µM Folic Acid Temperature dependent Emission Ex 360 nm

2.5

0.5 415 435 455 475 495 515

Wavelength (nm)

Folic acid (3µM) was excited at 360 nm under various temperatures (4, 14, 24, 34

and 44 0C) and the peak was observed at 445 nm with the intensity of 1.95 (Figure #11c).

Intensity level was decreased when the temperature was increased.

29

345 nm

Inte

ns

ity

Figure #11 d

Binary System (3µM Folic +0.7µM DHPR) Temperature dependent

150 Emission Ex 280nm 4C 120 14C

90 24C

60 34C

30 44C

0 290 340 390 440 490

Wavelength (nm)

Figure eleven (d) illustrates the temperature dependent emission at 280nm

for binary folic acid (3µM folic acid +0.7µM DHPR). Peak was observed at 445

nm with the intensity of 110. Decrease in intensity was observed with the increase

in temperature.

30

5

445 nm

Inte

ns

ity

Figure #11e

Binary System (3µM Folic +0.7µM DHPR) Temperature dependent Emission Ex 340 nm

4 4C

14C 3 24C

34C 2 44C

1

0 385 435 485 535 585

Wavelength (nm)

Figure eleven (e) shows the temperature dependent emission at

340nm for binary folic acid (3µM folic acid +0.7µM DHPR). Peak was observed

at 445 nm with the intensity of 3.99. Decrease in intensity was observed with the

increase in temperature.

31

5

445 nm

Figure #11 f In

ten

sit

y

Binary System (3µM Folic+0.7µM DHPR) Temperature dependent Emission Ex 360 nm

4 4C 14C

3 24C 34C

2 44C

1

0 385 435 485 535 585

Wavelength (nm)

Figure eleven (f) illustrates the temperature dependent emission at

360 nm for binary folic acid (3µM folic acid +0.7µM DHPR). Peak was observed

at 445 nm with the intensity of 4.35. Decrease in intensity was observed with the

increase in temperature.

32

345 nm

Inte

ns

ity

30

20

Figure #11g

TERNARY (3uM FOLIC+0.7uM DHPR+3uM NADH) EMISSION EX at 280

TEMP DEP 50 4C

40 14C

24C

34C 10

0 290 340 390 440 490

Wavelength (nm)

Figure eleven (g) shows the temperature dependent emission at 280nm for

ternary folic acid (3µM folic acid +0.7µM DHPR + 3µM NADH). Peak was

observed at 345 nm with the intensity of 34. Decrease in intensity was observed

with the increase in temperature.

33

3

Figure #11 h

Ternary System (3µM Folic+0.7µM DHPR+3µM NADH) Temperature dependent Emission

Ex 340 nm

Inte

nsi

ty

4C

2 14C

24C

34C

44C 1

0 360 410 460 510 560

Wavelength (nm)

Ternary folic acid (3µM folic acid +0.7µM DHPR + 3µM

NADH) was excited at 340 nm under various temperatures (4, 14, 24, 34 and 44 0C) and

the peak was observed at 445 nm with the intensity of 2.15 (Figure #11 h). Intensity

level was decreased when the temperature was increased.

34

445 nm

Figure #11 i

Ternary System (3µM Folic+0.7µM DHPR+3µM NADH) Temperature dependent Emission

Ex 360 nm

2.5

4C 2 14C

Inte

ns

ity

1.5 24C

34C 1

44C

0.5

0 380 430 480 530 580

Wavelength (nm)

Ternary folic acid (3µM folic acid +0.7µM DHPR + 3µM

NADH) was excited at 360 nm under various temperatures (4, 14, 24, 34 and 44 0C) and

the peak was observed at 445 nm with the intensity of 2.15 (Figure #11 i). Intensity level

was decreased when the temperature was increased.

280 nm

Inte

nsi

ty

35

Figure #12a

4

3

2

3µM Folic Acid Temperature dependent Excitation 360 nm

Em 345 nm 4C

14C

24C

34C

44C

1

0 250 260 270 280

Wavelength (nm) 290 300

Figure #12 b

5

4.5

4

3.5

3µM folic acid excitation EM @ 445 nm temperature dependent

4C

Inte

nsity

14C

34C

3

2.5

44C 2

1.5

1

0.5

0 235 255 275 295 315 335 355 375

Wavelength (nm)

Folic acid (3µM) was emitted at 345 nm (Figure #12a) and at 445 nm

(Figure #12 b) and has a peak at 280 nm and at 360 nm respectively.

36

Figure #12 c

BINARY (3µM FOLIC+0.7µM DHPR) EXCITATION EM @ 345nm TEMPERATURE DEPENDENT

350

300

280 nm

250

4C 200

14C 24C 34C 150 44C

100

50

0 200 220 240 260 280 300 320 340

Wavelength (nm)

Binary folic acid (3µM folic acid +0.7µM DHPR) temperature dependent

was emitted at 345 nm and reveals a peak at 280 nm.

Inte

ns

ity

37

Figure #12 d

280 nm BINARY (3µM FOLIC+0.7µM DHPR) EXCITATION EM @ 445 nm TEMPERATURE DEPENDENT

7

6

5 345 nm 14C

Inte

ns

ity

4

3

24C

34C

44C

2

1

0 240 290 340 390 440

Wavelength (nm)

Binary folic acid (3µM folic acid +0.7µM DHPR)

temperature dependent was emitted at 445 nm and reveals peaks at 280 nm and 345 nm.

38

345 nm

Inte

nsity

Figure #12 e

TERNARY (3µM FOLIC+0.7µM DHPR+3µM NADH) EXCITATION EM @ 345 TEMP DEP

Inte

nsity

280 nm

120

100

4C 80

14C

24C 60

34C

40

20

0 200 220 240 260 280 300 320 340

Wavelength (nm)

Figure #12 f

Ternary System (3mM Folic+0.7mM DHPR+3mM NADH) Excitation

Em 445 nm

5

4C 4

3

2

14C

24C

34C

44C

1

0 300 320 340 360 380 400 420 440 Wavelength (nm)

Ternary folic acid (3µM folic acid +0.7µM DHPR + 3µM NADH) was emitted at

345 nm (Figure #12 e) and at 445 nm (Figure #12 f) and has a peak at 280 nm and at

345 nm respectively.

Various concentration of solvent subtractedfolic acid in Infrared (IR) [1800-1200 cm-1]

39

Chapter 6

Folic acid in IR

Various concentrations of folic acid (100, 50, 25, 10, 5 and 1mM) with

the pH of 7.40 were prepared and ran in IR in order to identify their IR bands. Then, calculated

simulations were performed to folic acid using Gaussian 03 program to compare and contrast

with the experimental result. Folic acid was protonated with the help of Gaussian 03 program

and then subtracted from calculated simulation of folic acid to observe the changes in IR bands.

These IR spectra are shown in figure 13a, b, c, d, e, f, g, and h.

Figure #13a

1690 cm-1

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

-0.2 1270 1370 1470 1570 1670 1770

Wavenumber (cm -1)

100 mM fol50 mM foli25 mM fol10 mM foli5 mM folic ac1 mM folic ac

1600 cm-1

1480 cm-1

1450 cm-1

1400 cm-1

1340 cm-1

ic acid c acid ic acid c acid

id id

Ab

so

rba

nc

e

Various concentraions of solvent subtracted folic acidin Infrared (IR) [1100-750 cm-1]

40

Figure #13b

Ab

so

rb

an

ce

0.3

0.3

0.2

0.2

0.1

0.1

0.0

1070 cm-1

950 cm-1

910 cm-1

841 cm-1 814 cm-1

750 800 850 900 950 1000 1050 1100

Wavenumber (cm -1)

100 mM folic acid

50 mM folic acid

25 mM folic acid

10 mM folic acid

5 mM folic acid

1 mM folic acid

Various concentrations of folic acid were run in infrared

and IR peaks were identified. Observed a higher IR signal for higher concentration of

folic acid. Regions of 1800-1200 cm-1 and 1100-750 cm-1are shown in figures 13(a) and

(b) respectively.

41

-1980 cm 1 950 cm

Calculated simulations for folic acid

Figure #13c

Gaussian 03 Simulated Infrared (IR) for Folic Acid

Ab

so

rb

an

ce

[750-1100 cm-1] 70

60

50

40

30

20

10

0 750 850 950 1050

Wavenumber (cm -1 )

NH2 N

N

N

N NH2

N H

C O

N H

COOH

COOH

42

1670 cm -1

Figure #13d 1550 cm-1

Gaussian 03 Simulated Infrared (IR) for Folic Acid Region of [1200-1800 cm-1]

0

100

200

300

400

500

600

700

800

900

1000

Ab

so

rban

ce

1220 cm -1

1310 cm-1

1500 cm -1

N

N

N

N NH2

NH2

N H

C O

N H

COOH

COOH

1200 1300 1400 1500 1600 1700 1800

Wavenumber (cm-1)

Calculated simulations for folic acid was run in Gaussian 03 and

the IR spectra was obtained for the regions of 1800-1200 cm-1 and 1100-750 cm-1 were

presented in figures (d) and (c) respectively.

43

980 cm 1030 cm 880 cm-1 770 cm-1

940 cm-1

Figure #13e

Gaussian 03 Simulated infrared (IR) for protonated Folic Acid

[750-1100 cm-1]

Ab

so

rba

nc

e

200

180

160

140

120

100

80

60

40

20

0 750 800 850 900 950 1000 1050 1100

Wavenumber (cm-1)

N

N

N

N

NH3

NH3

N H

C

O

N H

COOH

COOH

44

1550 cm-1

Figure #13f

Gaussian 03 Simulated infrared (IR) for protonated Folic 1670 cm -1 Acid

[1200-1800 cm-1]

0

100

200

300

400

500

600

700

800

Ab

so

rban

ce

1220 cm -1 1310 cm -1

1410 cm -1

1730 cm -1

N

N

N

N

NH3

NH3

N H

C

O

N H

COOH

COOH

1200 1300 1400 1500 1600 1700 1800

Wavenumber (cm-1)

Calculated simulations for protonated folic acid was run in

Gaussian 03 and the IR spectra was obtained for the regions of 1800-1200 cm -1 (Figure

#13f) and 1100-750 cm-1 (Figure #13e) and IR peaks were identified.

45

980 cm 940 cm 1030 cm 1

Figure# 13g

Subtraction of Folic acid from the protonated Folic acid [750-1100 cm -1]

-1 Wavenumber(cm ) -20

0

20

40

60

80

100

120

140

160

180

750 800 850 900 950 1000 1050 1100

Ab

so

rba

nc

e

46 1400 cm-1

1540 cm-1 1590 cm-1

Figure #13h

Subtraction of Folic acid from the protonated Folic acid [1200-1800 cm -1]

-620

-520

-420

-320

-220

-120

-20

80

1200 1300 1400 1500 1600 1700 1800

Wavenumber(cm -1)

Ab

sorb

ance

1500 cm-1

1660 cm-1

Resultant (protonated – folic acid) calculated simulations

for folic acid with the regions of 1800-1200 cm-1 and 1100-750 cm-1 and identified IR

peaks were shown in figure 13 (h) and (g) respectively.

47

Chapter 7

Conclusions

Our main goal of this research is to study the interaction between protein structure

Dihydropteridine Reductase, cofactor Nicotinamide Adenine Dinucleotide (NADH), and

the inhibitor folic acid and thus we studied them using spectroscopy technique. Our

excitation spectra revealed the peak at 280 nm and at 360nm as it was in the UV spectra.

As a result, 280 nm and the 360 nm peaks are real because according to the theory if the

UV peaks are seen in the excitation spectra then those peaks are real. Our fluorescence

spectroscopy spectra show the intensity difference within folic acid (3µM) by it self,

binary (3µM folic acid + 0.7µM DHPR), and ternary (3µM folic acid + 0.7µM DHPR +

3µM NADH) systems. These intensity changes exhibit the interaction of protein-ligand

interaction. At the same time, we also observed the decrease in intensity level as the

temperature was raised and indicating the inverse relationship with the fluorescence

intensity and the temperature.

We ran the folic acid in IR to study the nature of the interaction. Various

concentrations of folic acid were prepared and ran in IR as it was in figures 13a and b.

Then, calculated simulations for folic acid was performed in Gaussian 03 to compare the

IR data with the experimental data. Similar IR bands were observed in both calculated

simulation and in experimental spectra. However, the minor differences in these IR bands

are due to the fact that Gaussian calculations were done in vacuum while the experiments

were carried out in tris HCl solvent. At the same time, folic acid structure was protonated

in one and three position using Gaussian and subtracted from non-protonated folic acid to

see which bonds are moving upon protein binding (figures 13 g and h).

48

References

1Spectroscopy – UV/Vis. http://www.sci.sdsu.edu/TFrey/Bio750/UV-VisSpectroscopy.html. Retrieved on April 4th 2007.

2Fluorometers.http://www.iugaza.edu/users/abdellatif/Instrumental%20Analysis/Lectures /Lectures%2021-/L30.pdf . Retrieved on March 20th 2007.

3 Jablonski Diagram. Research\Jablonski Diagram.htm. Retrieved on April 6 th 2007.

4 Infrared Spectroscopy. http://sis.bris.ac.uk/~sd9319/spec/IR.htm. Retrieved on April 10th 2007.

5 Higher Education Commission. http://www.hec.gov.pk/htmls/thesis/thesis_detail.asp?op=72. Retrieved on April 20th 2007.

Joseph R., Lakowicz. (1992). Topics in fluorescence Spectroscopy. New York: Plenum Press.

Guilbault, George G. (1973). Practical fluorescence; theory, methods, and techniques. New York, M. Dekker.

Rao, C. N. R. (1967). Ultra-violet and visible Spectroscopy; chemical applications. New York, Plenum Press.

Szymanski, Herman A. (1964). IR, theory and practice of infrared Spectroscopy. New York, Plenum Press.