This document discusses the binding of Pyroloquinoline Quinone (PQQ) to Human Serum Albumin (HSA). UV-Vis spectroscopy shows the absorption of HSA increases with the addition of PQQ, indicating their binding. Fluorescence spectroscopy and quenching experiments determine the binding constant between PQQ and HSA is 2.194 × 107 M-1. Circular dichroism experiments reveal PQQ causes a loss of α-helical structure in HSA. Molecular docking simulations identify the highest affinity binding site of PQQ on HSA is site I, and hydrogen bonding stabilizes their complex formation.

1. 1

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
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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
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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
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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
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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
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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
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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
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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
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11. Active site amino acid residues of PQQ at site I
of HSA
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12. A)
B)
A) RMSD plot of HSA- PQQ
system
B) Number of Hydrogen
bonds
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13. hydrogen bonding (pink arrow) and salt bridges interactions of PQQ
with respective residues
(Lys199, LYS 195, SER192, TYR150)
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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
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15. THANK YOU.
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