PROTEIN-LIGAND INTERACTION Molecular recognition refers to the process in which biological macromolecules interact with each other or with various small molecules through non-covalent interactions to form a specific complex. Here we will discuss the interaction between hemoglobin protein and bovine serum albumin with their respective ligands. Hemoglobin oxygen carrying capacity and hemoglobin dissociation curve will also be discussed. UV titration, fluorescence quenching, FRET and molecular docking experiments will also be performed to determine the binding interactions between proteins and ligands.  The specific ligand will bind to specific active site of specific protein. This active site provides a region for ligand to interact with protein by forming a protein- ligand complex or PL complex. This complex will help various biological process to occur and when the biological process is complete, and product is formed then the protein and ligand separates individually.

1. PROTEIN-
LIGAND
INTERACTION
B Y K H U S H I VA S U D E V

2. ABSTRACT
• Molecular recognition refers to the process in which biological macromolecules interact with each other or
with various small molecules through non-covalent interactions to form a specific complex.
• Here we will discuss the interaction between hemoglobin protein and bovine serum albumin withtheir
respective ligands. Hemoglobin oxygen carrying capacity and hemoglobin dissociation curve will also be
discussed.
• UV titration, fluorescence quenching, FRET and molecular docking experiments will also be performed to
determine the binding interactions between proteinsand ligands.

3. INTRODUCTION
• The specific ligand will bind to specific active site of specific protein. This active site provides a region for
ligand to interact with protein by forming a protein- ligand complex or PL complex. This complex will help
various biological process to occur and when the biological process is complete, and product is formed then
the protein and ligand separates individually.

4. HAEMOGLOBIN
• It is an oxygen carrying pigment which is present in the Red Blood Cells. The red color of blood is due to
the presence of Hemoglobin. It is a protein molecule and known for performing multiple biological functions,
such as oxygen transportation during respiration from the lungs to every tissue of the body and bring back
carbon-di-oxide from tissue to the lungs. Hemoglobin develops in the bones in the red bone marrow. It is
made up of the goblin protein and the iron rich compound heme. Each hemoglobin can bind to 4 oxygen
molecules at a time. The molecular weight of hemoglobin is 65000. Red blood cells lack in nucleus for
increasing the surface area for oxygen binding. The oxygen carrying capacity of Hb is 1.34 mlO2/g. in 100 ml
of blood, there is about 15 15gm of Hb so that 100 ml of blood has the capacity to bind 20.1ml of oxygen.
This is called the oxygen binding capacity ofblood.

5. STRUCTURE
• The structure of hemoglobin is a combination of heme, and globin protein arranged through
coordinated bonds. Globin protein consists of four polypeptide chains. Two of them are of
alpha type and two of them are of beta type thus, it is also known as alpha2 beta2 type. one
hundred and forty-one amino acids are present in each alpha chain. One hundred and forty-
six amino acids are present on each beta chain. Each polypeptide chain forms a cup like
structure with a pocket like area which fits the prosthetic group, heme is buried. Heme had
iron which I linked to the imidazole nitrogen of the histidine in position 58 and 87 amino acid
of alpha chain. Each iron which is present at every centre of the polypeptide chains is linked
with four nitrogen’s of each pyrrole ring.
• During oxygenation, the arrangement of subunits is altered. The beta chains appear closer
together and their heme groups are closer by 7 Å upon oxygenation.

6. EXPERIMENTAL SECTION
1.CONCENTRATION DETERMINATION
2. UV TITRATION
3.MOLECULAR DOCKING
4.FLORESCENCE QUENCHING
5.CIRCULAR DICHROISM

7. 1.CONCENTRATION
DETERMINATION
AIM-: TO DETERMINE CONCENTRATION OF HEMOGLOBIN IN PBS
MATERIAL REQUIRED-: PBS (7.4ph), BSA, Cuvette, pipette (1ml),pipette tip.
Software required-: Aspect UV
Instrument-: UV-VISIBLE SPECTROMETER
Procedure
1. Sterilize all the equipment and cuvette with ethanol.
2. With the help of 1 microlite pipette, load 3ml PBS in each cuvette and put them in
their holder which is located in the uv-visible spectrometer.

8. 3.Now click to open Aspect UV software and open module icon and click further on spectrum.
4.Now click on reference and a graph will be obtained where 100% transmittance will be observed.
NOTE: 100% transmittance is observed because there was no sample present in the cuvette.
Thus, 0% absorbance.
5.after this load 3ml haemoglobin in sample cuvette and load PBS in the reference cuvette.
6.Click on measure and set absorbance range 0-1.
Observation
• The peak observed at 408nm having absorbance 0.79.
wavelength 408nm 409nm 410nm 411nm 412nm
absorbance 0.7913 0.7865 0.7739 0.7548 0.7285

9. CALCULATION
Calculation-:
According to Lambert
law,
•A = εlc
Given values-:
A=0.7913
EPSILON=12280
LENGTH= 1cm
Concentration=?
A = εlc
0.71=12280*1*C 0.71/12280=C
57.81micromolar=C

10. OBSERVATION
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250 300 350 400 450 500 550 600
WAVELENGTH(nm)

11. UV TITRATION
AIM-: TO DETERMINETHE ABSORBANCEOF HAEMOGLOBIN-PHOSMET BY UV-TITRTION.
MATERIAL REQUIRED-: Haemoglobin, 5ml Phosmet(1mg/ml) etc INSTRUMENT-: UV-VISIBLE SPECTROMETER
Procedure-:
1. Sterilize all the equipment and cuvette with ethanol.
2. With the help of 1 microliter pipette, load 3ml PBS in each cuvette and put them in their holder which is located in the uv-visible spectrometer.
3.Now click to open Aspect UV software and open module icon and click further on spectrum.
4.Now click on reference and a graph will be obtained where 100% transmittance will be observed.
NOTE: 100% transmittance is observed because there was no sample present in the cuvette. Thus, 0% absorbance.

12. 5. after this load 3ml Hemoglobin in sample cuvette and load PBS inthe
reference curette.
6. Click on measure and set absorbance range 0-1.
7.Add 10microlitre Phosmet solution to the already kept Hemoglobin
cuvette. 1 PBS Hemoglobin 0 0
2 PBS Sample1+
Phosmet
10 10
3 PBS Sample2+
Phosmet
10 20
4 PBS Sample3+
Phosmet
20 40
5 PBS Sample4+
PHOSMET
20 60
6 PBS Sample5+
Phosmet
20 80
7 PBS Sample6+
Phosmet
20 100
8 PBS Sample7+
Phosmet
20 120
9 PBS Sample8+
Phosmet
20 140
10 PBS Sample9+
Phosmet
20 160
Observation-: It was observed that when we were adding
PHOSMET in the HEMOGLOBIN, the absorbance peak
also increasing due to HEMOGLOBIN-PHOSMET
interaction.
Sampl
eno.
reference contents Amount
of
Phosmet
added(µl)
Total
amount of
Phosmet
in the
sample(µl)

13. • Observation-: It was observed that
when we were adding PHOSMET in
the HEMOGLOBIN, the absorbance
peak also increasing due to
HEMOGLOBIN-PHOSMET interaction.

14. MOLECULAR DOCKING
Aim -: To determine haemoglobin-phosmet interaction by molecular docking.
Material required-: Schrödinger software is required.
Theory -: Schrödinger software has three tools such as maestro, maestro element and material science or
studio. This software can’t be use by anyone because license is required which is provided through software
company and then software company will ensure your work through the respective scientist. And at last, they
provide license. Mainly for protein-ligand interaction maestro tool is used.
Procedure-:
1. Maestro tool is clicked and opened.
2. Now click on file and select import structure function.
3. And then download your 3Dprotein structure by entering PDB ID.PROTEIN PREPARATION

15. 4. Select protein preparation function then clicks on review and modifyand select analyze workplace option.
5. Eliminate unwanted chains expect the chain having ligand.
6. Now to view any sought of ligand binded with protein by default.You can simply change the style of
the ligand.
7. And then if those ligands are not required then select right click toeliminate them as well. Now
unpaired bond will be visible.
8. now click on reprocess icon and then select on generate state forHetero atom process.
9.After this click on refine function and then do the following such asoptimize, remove water, and
minimize.
LIGAND PREPRATION
10. Now download ligand structure from import in .sdf format.

16. 11. after this click on task icon and select ligprep function.
12. And select workplace option and
allow to run.DOCKING
13. Now select receptor grid generation for protein-ligand
interaction.
Observation-:
Docking score is obtained for specific protein-ligand
interaction.

17. FLORESENCE QUENCHING
• Aim -:
• Material Required-: cuvette(3ml), BSA(7.4ph), phosmet, hemoglobin, pipette, pipette tip etc.
• Instrument-: FLORESCENCE SPECTROPHOTOMETER
• Theory-: It is a phenomenon in which the florescence intensity of light emitting molecule is decreased. When the
intensity of given molecule is decrease in the presence of another molecule This phenomenon will be termed as
florescence quenching. A variety of molecular interactions can result in quenching. These include excited- state reactions,
molecular rearrangements, energy transfer, ground- state complex formation, and collisional quenching.
• QUENCHER-
• It is the substance or molecule which will decrease the intensity of given sample. It is also known as the quenching
agent.

18. • PROCEDURE
1. First open the florescence software.
2. Then select the excitation function and set wavelength (295nm) and slit (2.5).
3. After this click on emission and set emission 300-450, slit=2.5 and accessory will bewater jacketed single sell.
4. Now pour 3ml of Haemoglobin through pipette in the florescence cuvette.
5. Then fix the cuvette in the florescence spectrophotometer.
6. And then allow the device to run and obtain the graph.
7. After this add PHOSMET in the following manner.
1. Hemoglobin
2. Hemoglobin+20SGO
3. Hemoglobin+40SGO
4. Hemoglobin+60SGO
5. Hemoglobin+80SGO
6. Hemoglobin+100SGO
7. Hemoglobin+120SGO
8. Hemoglobin+140SGO
9. Hemoglobin+160SGO

19. 8. Now discard the sample from cuvette and then add fresh
Haemoglobin in the cuvette.
9. after this select synchronous option with excitation
range 250nm-350nm, slit-5nm andemission will be 15nm.
10. then add PHOSMET in the following manner.
11. And then save the data and select new and select chiller at
temperature 30degree Celsius.
12. And repeat as it is by adding PHOSMET in the same
manner.
13. Now follow this again at 35 temperatures.
14. At last compile all the data to obtain the graph with the
help of origin software.
1. Hemoglobin
2. Hemoglobin+20SGO
3. Hemoglobin+40SGO
4. Hemoglobin+60SGO
5. Hemoglobin+80SGO
6. Hemoglobin+100SGO
7. Hemoglobin+120SGO
8. Hemoglobin+140SGO
9. Hemoglobin+160SGO

20. OBSERVATION

21. 1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
0 0.00005 0.0001 0.00015 0.0002
fº/f
[Phos] (M)
298.15 K
303.15 K
308.15 K
-1.60
-1.40
-1.20
-1.00
-0.80
-0.60
-0.40
-0.20
0.00
-5.00
-4.80
-4.60
-4.40
-4.20
-4.00
-3.80
Log
[(f0-f)/f]
Log [Phos] (M)
298.15 K
303.15 K
308.15 K
y = 28497x - 88.297
R² = 0.9553
0
2
4
6
8
10
12
0.0032 0.00325 0.0033 0.00335 0.0034 0.00345
2.303RLOG
K
1/T

22. CIRCULAR DICHROISM
• AIM-: TO DETERMINE SECONDARY STRUCTURE OF PROTEIN AND ITS CONFORMATION.
• MATERIAL REQUIRED-: cuvette, PBS, Hemoglobin, pipette, temperature controller, nitrogen gas cylinder.
• Device-:
• CD SPECTROPHOTOMETER
• SOFTWARE-:
• CD MANAGER
• THEORY-: Alpha-helix structure, β pleated sheet and other random coil are easily determined by CD
spectroscopy.CD manager software is common. Although other software can be used according to your
sample. Nitrogen cylinder is quite necessary for the device so that the light source can easily transmit
through the glass side of cuvette

23. • PROCEDURE-:
1. Firstly, open nitrogen gas cylinder which is directly attached to the CD spectrophotometer.
2. Then wait for couple of minutes for the continuous flow of nitrogen gas.
3. After this “ON” your device and after two minutes load PBS in the cuvette.
4. And initialize the measurement by naming the cuvette cell.
5. After this load sample in the cuvette and initialize the measurement by opening the CD
spectrophotometer software.
• Then close lid of device and obtain the graph.

24. OBSERVATION

25. CONCLUSION AND FUTURE
SCOPE
• Through this we can conclude that protein shape induced when ligand
bind to its specific protein and as the concentration increases then
ligand will mask the protein active site and fluorescence decreases.
Thus, protein ligand plays important role in the biochemical process.