E4

Bovine Serum Albumin (BSA) bioconjugation with Fluorescein isothiocyanate (FITC) and
Fluorescein-5- Maleimide
• Bioconjugation consists in the linking of two or more molecules forming a novel complex with
combined properties of its individual starting components.
• Proteins, nucleic acids and other molecules can be labelled by small modifying agents, referred
as tags and probes.
• Serum albumins, in particular, are the most extensively studied and applied proteins because of
its availability, low cost, stability and unusual ligand-binding properties.
• Fluorescent tags can provide very high sensitivity exploiting their large quantum emission yield.
Each fluorophore has a chromogenic property and different reactive groups (for example amine-
reactive or thiol-reactive) which can couple to specific functional groups of the target molecules.
• Our presentation talk through the bioconjugation of a protein ( BSA-bovine serum albumin)
using two different fluorescent probe -fluorescein isothiocyanate (FITC) , fluorescein-5-
maleimide and the subsequent set on using absorbance spectroscopy.
FITC(Fluorescein
isothiocyanate )
•Fluorescein is one of the most popular among
fluorescent labelling agents or probes. In particularly,
fluorescein isothiocyanate is one of the most popular
fluorescent probe. Its fluorescent character is due to its
three-ring planar structure, high absorptivity, excellent
fluorescence quantum yield and good water solubility.
•Molar mass: 389.382 g/mol
•Formula : C21H11NO5S
•Melting point : 359.5 °C
•Density : 1.542 g/mL
Fluoresceine-5-maleimide
•Fluorescein-5-maleimide is the most widely used
green fluorescent thiol-reactive dye. The reaction
requires very mild conditions and is highly specific.
•Thiols are good nucleophiles, even at neutral pH,
and have the added benefit of scarcity in proteins
and other biomolecules. Common reagents for
modifying thiol groups are maleimides, alkyl
halides and iodoacetamides, and activated
disulfides.
•Formula: C24H13NO7
•Molecular Weight: 427.4g/mol
Molar Extinction
Coefficient
Its a measure of how strongly a chemical
species or substance absorbs light at a
particular wavelength.

𝐴=𝜀 𝐶𝐿
Here, A, , C, L respectively Absorbance,
Molar attenuation coefficient,
concentration and path length.
 ASSERTAINMENT OF BSA EXTINCTION
COEFFICIENT FOR FITC:

BSA molar extinction coefficient in PBS

0.8

0.7
Volume Absorbance concentration
f(x) = 33426.4840230597 x + 0.00458859903812825
0.6 0 0 0
0.3 0.152 0.0000044
0.5 0.5 0.338 0.00000981
Absorbance

BSA 0.7 0.561 0.0000164

0.4 Linear
(BSA) 0.9 0.667 0.0000201
0.3

0.2
BSA 𝛆 experiment 𝛆 literature
0.1
unit M-1cm-1 M-1cm-1
0
0 0.000005 0.00001 0.000015 0.00002 0.000025 BSA 280nm 33426 38576
BSA mol/l
 ASSERTAINMENT OF FITC EXTINCTION
COEFFICENT:
FITC molar extinction co-efficient in PBS
1

0.9 f(x) = 45669.6590768185 x + 0.00512846587603233 Volume Absorbance Concentration

0 0 0
0.8
0.3 0.275 0.0000057
0.7 0.5 0.432 0.00000962
ABS

0.6 0.7 0.621 0.00001335

0.5 0.9 0.812 0.0000173
FITC 1.2 0.931 0.0000206
0.4 Linear
0.3
(FITC)
FITC 𝛆 experiment 𝛆 literature
0.2 Unit M-1cm-1 M-1cm-1
0.1
FITC 45670 62348
0
0 0.000005 0.00001 0.000015 0.00002 0.000025

FITC mol/l

Comments- Not properly satisfying

with the literature value, maybe
some drawbacks in the preparation
of the standard solution.
ASSERTAINMENT OF BSA EXTINCTION COEFFICIENT FOR
MALEIMIDE :
BSA extinction co-efficient in PBS
0.8
Volume Absorbance Concentration
0.7
0.67
0 0 0
0.6 f(x) = 32236.5776667028 x − 0.00634337069570012
0.3 0.152 0.0000044
0.5 0.501
BSA 0.5 0.28 0.00000981
0.4
ABS

Linear
(BSA) 0.7 0.501 0.0000164
0.3 0.28
0.9 0.67 0.0000201
0.2
0.152
0.1 BSA 𝛆 experiment 𝛆 literature
00
0 0.000005 0.00001 0.000015
BSA mol/l
0.00002 0.000025 unit M-1cm-1 M-1cm-1

BSA 280nm 32237 38576

ASSERTAINMENT OF F-5-MALEIMIDE EXTINCTION
COEFFICENT
Maleimide molar extinction co-efficient
0.9
Volume Absorbance Concentration
0.8
f(x) = 36920.5316944617 x + 0.0225673112742841
0.7 0 0 0
0.6
0.18 0.09 0.00000121
0.5
0.3 0.286 0.00000684
ABS

0.4

0.3 0.5 0.449 0.0000114

0.2 0.7 0.616 0.00001597

0.1
0.9 0.769 0.0000205
0
0 0.000005 0.00001 0.000015 0.00002 0.000025

Maleimide mol/l

Comments- Fluorescein 5 maleimide groups are moisture sensitive also it must

have some –SH groups available(thiol) and may oxidize and create disulphide
bonds which can not react with maleimide.
BIOCONJUGATION PROTOCOL-GEL FORMATION

• Use Sephadex G-25 column , PBS buffer as eluent.

• Equilibrium of the packed bed with buffer,
bioconjugate is eluted isocratically, by gravity and
different fractions collected.
• Smaller sized molecules enter the beads in the
column while large molecules are excluded and
leave the column first, followed by smaller
molecules.
• Sephadex G-25 has a fractionation range for
globular proteins of 1000 to 5000 g/mol molecular
weights, with an exclusion limit of approximately
5000 g/mol. This means that proteins and peptides
larger than 5000 g/mol are therefore easily
separated from molecules with molecular weigth
of less than 1000 g/mol, which is the case of BSA-
FITC and FITC. Fig. Indicative sketch of gel filtration procedure
• Free FITC easily separated from the bioconjugate.
EVALUATION OF THE DYE/PROTEIN RATIO

• The dye/protein ratio (D/P) of the bioconjugate can be determined by the absorption spectra of the labelled proteins,
according to the relationship reported in equation,

D/P
Where C is the correction factor, valued 0.29

D/P(FITC): 1.206
D/P MALEIMIDE: 1.78
Comments:
• Fluorescent dyes are hydrophobic and may bind non covalently to proteins.
For accurate information of the Dye : protein molar ratios, extensive dialysis
must be performed to remove any non specifically bound dye.

• Free fluorophores must be completely removed, since their presence can

adversely affect the determination of labelling efficiency (D/P)
References
• Barbero, Nadia, Claudia Barolo, and Guido Viscardi. "Bovine Serum Albumin Bioconjugation
with FITC." World Journal of Chemical Education 4, no. 4 (2016): 80-85.

• Hermanson, G. T. Bioconjugate Techniques, 2 nd edition, Academic Press, Elsevier, 2008

• Bigelow, D.J. and Inesi, G. (1991). Frequency-domain fluorescence spectroscopy resolves the
location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-
ATPase of sarcoplasmic reticulum. Biochem. 30:2113-25

• https://it.wikipedia.org/wiki/Isotiocianato_di_fluoresceina