Vibrational Spectroscopic Study of Biological Systems
Rekha
a
Gautam ,
aDepartment
Bhagwat
b
Chandrasekar ,
Srabanti
b
Rakshit ,
Dipankar
b
Nandi ,
U.
c
Nongthomba
and Siva
a
Umapathy *
of Inorganic and Physical Chemistry, bDepartment of Biochemistry, cDepartment of Molecular Reproduction Development and Genetics
Indian Institute of Science, Bangalore-560012, India.
Why Drosophila?? IFM (Indirect Flight Muscles)
Muscle-Related Disorders
APAP most widely used analgesic and
antipyretic agent in the world
Hepatotoxicity
Metabolic Activation of APAP
Liver: important for cellular
metabolism
Highly susceptible to drug toxicity
Musculoskeletal
Cardiomyopathies
Nemaline Myopathies
To investigate
 Short life span
 Structural similarities to vertebrate
skeletal muscles
 Available genome database
induced hepatotoxicity
 Dispensable to flies
 60% of human disease genes
have counter parts in Drosophila
Dorsal Longitudinal
Muscles
Drosophila melanogaster
 Conservation of developmental
pathways
Troponin-T mutants in Drosophila
Most Popular Drug
 All the developmental stages are well
characterised
Lead Cause of Acute Liver Failure
up101
up1
control
BALB/c-mice starved overnight;
 Mice dosed with water
 dosed with APAP
 Mice dosed with APAP and L-methionine
C
b
c
e
f
6hr
3000
3200
3400
3600
1.5hr
2800
Wavenumber (cm-1)
Glycogen Levels
(1030/1080)
1080
up101
1hr
1
up1
0.95
1.1
1.05
control
0.9
1
0.85
0.95
0.8
0.9
set1
set2
set3
1661
1000
1200
1400
1600
3000
3200 3400
Wavenumber (cm-1)
3600
0.85
0.75
Control
1.5hr
3hr
6hr
Control 1.5hr
3hr
6hr
After 6hr
control
1000
1050
1100
1150
1200
2850
(cm-1)
2900
2950
CH2/CH3 with injury
3000
Wavenumber (cm-1)
1200
1300
1400
1500
1600
Increase in Lipid/Protein
1700
861
944
Conclusions
1100
1.15
Raman Spetrum of Serum
Wavenumber
1000
3600
Ester bonds 1171/1153
set1
set2
set3
1030/1080 with injury
900
3400
1.2
IR Image @ amide I
950
800
3200
1.1
1171/1153 with injury
Reverse Kinetics Study of liver injury
Raman Shift cm-1
0.534
up1
control
0.524
Change in secondary structure of proteins
α-helix to β-sheet / Random Coil
APAP
Post_Lmet
Pre_Lmet
Control
APAP
up101
600
Post_Lmet
Decreasing
References
0.484
α-helix
1625
1650
1675
Raman Shift cm-1
1700
β-sheet
1.
Bas W. D. de Jong, Tom C. Bakker Schut, Kees Maquelin, Theo van der Kwast,
Chris H. Bangma,|Dirk-Jan Kok, and Gerwin J. Puppels Anal. Chem. 2006,
78, 7761-7769.
2.
Christoph Krafft , Daniela Codrich ,Gloria Pelizzo , Valter Sergo
Vibrational Spectroscopy 2008),46,141–149.
3.
L. Chiriboga, P. Xie, H. Yee, V. VIigorita, D. Zarou, D. Zakim, M. Diem
Biospectroscopy, 1998, 4 , 47–53 .
Pre_Lmet
up101
1400
1600
2800
Raman Shift cm-1
2900
3000
3100
References
1. N.A. Nikonenko, D.K. Buslov, N.I. Sushko, R.G. Zhbankov Journal of Molecular
Structure 2005, 752 20–24.
Control
up1
1200
Change in Lipids due to lipid peroxidation
0.474
control
1000
Decrease in Glycogen
0.504
0.494
800
Conclusions
Increase in Glycogen levels
0.514
I1657 / I1667
700
1600
3000
Wavenumber (cm-1)
1.05
2925
up101
up1
control
1400
Glycogen level 1030/1080
0.5hr
C-H Stretch
1200
A Noninvasive Approach
Serum
control
1030
1298
1080
1034
940
778
1600
upheld101 (E88K)
Control Vs. Mutant of IFM
up101
up1
control
1400
2852
upheld1(splice mutant)
Canton-S
1200
1000
6hr
1.5hr
1hr
0.5hr
control
Decreasing
1000
Control
Kinetics Study of liver injury
d
600mg/kg
400mg/kg
200mg/kg
Control
Decreasing
B
a
Dose Dependent Study
APAP
C-H
Stretch
A
Excessive consumption leads to hepatotoxicity
Methods and Materials
FTIR Control Vs. Mutant of IFM
Glycogen
Americans take over 8 billion pills
of Tylenol (APAP) per year
600mg/kg
 Large number of eggs
APAP (Paracetamol)
400mg/kg
Account for roughly 10% of all the
known diseases in India
N-acetyl-para-aminophenol
200mg/kg
Neuromuscular
Parkinson’s Disease
IR Image @ amide I
1000
1200
1400
1600
3000
3200
Wavenumber (cm-1)
3400
3600
2.
Christoph Krafft , Daniela Codrich ,Gloria Pelizzo , Valter Sergo Vibrational
Spectroscopy 2008),46,141–149.
Acknowledgement: Department of Biotechnology (DBT), Department of Science and Technology (DST) , Council of Scientific and Industrial Research (CSIR), Indian Institute of Science (IISc)