2. Human Serum Albumin
• 585 amino acid residues
• Synthesised and secreted from the liver
• Maintain the oncotic pressure of blood
• Serves as a transport vehicle
• Molecular weight of HSA is roughly 66,700 Daltons
• C2936H4624N786O889S41
• Extraordinary ligand binding capacity
2
3. STRUCTURE
• One N-terminus, one C-
terminus and three
homologous domains,
named domain I,
domain II and domain
III
• Subdomains contains 4
to 6 α–helices
• 17 intramolecular
disulfide bridges that
stabilizes the HSA
3
4. Pyroloquinoline Quinone
• C14H6N2O8
• Methoxatin
• Red, highly polar, acidic
compound
• Characteristic green
fluorescence
• Absorption band between
300 and 420 nm
4, 5-dihydro(4), 5-dioxo(1)H-
pyrroloquinoline-2,7, 9
tricarboxylic acid
4
5. UV Spectra of HSA (10 µM) with addition of PQQ (0-3µM)
200 250 300 350 400
0.0
0.5
1.0
1.5
2.0
2.5
Absorbance
wavelength(nm)
• Two absorption peaks at 221 and 280 nm
• UV absorption intensity of HSA increased with gradual
addition of PQQ , hyperchromic shift
5
6. Fluorescence quenching of HSA (10 µM) with addition of PQQ (0-6 µM)
•With the incremental
addition of PQQ to
HSA, the fluorescence
intensity of HSA
decreased regularly
6
7. -6.0 -5.8 -5.6 -5.4 -5.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
log
(Fo/F)
log(Q)
Logarithmic plots of HSA with addition of PQQ
Stern-Volmer equation is used to estimate the binding constant (K) and binding
number (n)
log (F0-F)/F = log Kb + nlog[Q]
Kb = 2.194 × 107 M-1
7
8.
• Tryptophan and Tyrosine
• Conformational change
• Fluorescence quenching of HSA
upon addition of PQQ
• Indicates strong binding of PQQ
with HSA molecules 8
9. 200 210 220 230 240
-200
-150
-100
-50
0
Ellipticity
Wavelength (nm)
HSA
0.5 equivalent of PQQ
1 equivalent of PQQ
Circular Dichroism
The CD spectra of HSA-PQQ
• Band intensity
decreased by 57%
• Loss in α-helix
content
• Increase in
disorderness of
HSA
9
10. Representation of docking results showing PQQ at site I of HSA
Site I -10.17 kJ mol-1
Site II -6.29 kJ mol-1
Site III -4.14 kJ mol-1
10
13. hydrogen bonding (pink arrow) and salt bridges interactions of PQQ
with respective residues
(Lys199, LYS 195, SER192, TYR150)
13
14. • The drug(PQQ) interacts with HSA through complex
formation which was confirmed from UV-Vis spectra analysis
• The fluorescence results showed that PQQ acts as a strong
quencher
• Binding constant was found to be 2.194 × 107 L mol-1
• Tyrosine residue in the ligand binding site supported the
synchronous fluorescence quenching results
• PQQ binds to site I of HSA and formed a stable complex by
hydrogen bonds which was depicted from molecular docking
studies
• Molecular dynamics study shows the HSA-PQQ complex was
relatively stable throughout the whole simulation period
14
