BSA-FITC(Bovine Serum Albumin Bioconjugation with FITC)

Bioconjugation [1] consists in the linking of two or more molecules forming a novel complex with combined properties of its individual starting components. Thus, a protein can be labelled to another molecule capable of being detected to form a traceable conjugate. The resulting complex is thus visible, detectable and can be easily localized through various techniques (fluorescence spectroscopy, imaging), or directly used for measurements. Growing interest is caused by the possibility of applying a variety of bioconjugate techniques in biological, medicinal, polymer, material science or chemistry fields.

One of the key component for biological research and medicinal chemistry relies in the ability to produce a labelled protein with specificity for another target molecule. Proteins are therefore the most common targets for modification or conjugation techniques. Serum albumins, in particular, are the most extensively studied and applied proteins because of its availability, low cost, stability and unusual ligand-binding properties. [2] For this reason, a huge number of papers and reviews dealing with albumins have been published so far [<a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Barbero, N.; Barni, E.; Barolo, C.; Quagliotto, P.; Viscardi, G.; Napione, L.; Pavan, S.; Bussolino, F. A study of the interaction between fluorescein sodium salt and bovine serum albumin by steady-state fluorescence. Dyes Pigments 2009, 80 (3), 307-313.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference2″ name=”BRef2″>2, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Peters T. Serum albumin. Adv. Prot. Chem. 1985, 37, 161-245.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference3″ name=”BRef3″>3, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Carter, D. C.; Ho, J. X. Structure of serum albumin. Adv. Prot. Chem. 1994, 45, 153-203.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference4″ name=”BRef4″>4, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Barbero, N.; Visentin, S.; Viscardi, G. The different kinetic behaviour of two potential photosensitizers for PDT. J. Photochem. Photobiol. A: Chem. 2015, 299, 38-43.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference5″ name=”BRef5″>5] and above all for its low cost has been used for several laboratory experiments [<a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Williams, K.R.; Adhyaru, B.; Pierce, R.E.; Schulman S.G. The Binding Constant for Complexation of Bilirubin to Bovine Serum Albumin. An Experiment for the Biophysical Chemistry Laboratory. J. Chem. Educ. 2002, 79 (1), 115-116.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference6″ name=”BRef6″>6, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Liu, J. Investigation of Cu(II) Binding to Bovine Serum Albumin by Potentiometry with an Ion Selective Electrode: A Biophysical Chemistry Experiment for the Undergraduate Curriculum. J. Chem. Educ. 2004, 81 (3), 395-397.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference7″ name=”BRef7″>7, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Liang, P.; Adhyaru, B.; Pearson, W.L.; Williams K.R. The Binding Constant of Estradiol to Bovine Serum Albumin. An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting. J. Chem. Educ. 2006, 83 (2), 294-295.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference8″ name=”BRef8″>8, <a style="list-style-type: none; text-decoration: none; margin: 0px; padding: 0px; color: #fb3004; font-weight: normal;" title="Sohl, J. L.; Splittgerber, A. G. The binding of Coomassie brilliant blue to Bovine Serum Albumin: A physical biochemistry experiment. J. Chem. Educ., 1991, 68 (3), 262-264.” href=”http://pubs.sciepub.com/wjce/4/4/3/index.html#Reference9″ name=”BRef9″>9].

Proteins, nucleic acids and other molecules can be labelled by small modifying agents, referred as tags and probes. They usually contain atomic or molecular moieties with a sensitive detectability due to intrinsic chemical or atomic property such as fluorescence, radioactivity or bioaffinity toward another protein. A probe can contain a reactive group able to link to the functional groups of the biomolecule of interest. After the modification of a protein, the probe is covalently attached. In this way, the protein results permanently labelled with a unique detectable property.

Fluorescent tags [1] can provide very high sensitivity exploiting their large quantum emission yield. There are a great number of fluorophores available for a wide range of applications. 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. Fluorescein is one of the most popular among fluorescent labelling agents. In particularly, fluorescein isothiocyanate (FITC, Figure 1) is one of the most popular fluorescent probe. Its fluorescent character is due to its three-ring planar structure.

Isothiocyanates are reactive toward nucleophilic sites such as amines (i.e. lysines), sulfhydryls (i.e. cysteines) and the phenolate ion of tyrosine side chains [10]. However, FITC is highly selective for N-terminal amines in proteins [11] because it is able to form stable products only with primary amine groups. The reaction proceeds with the attack of the nucleophile to the electrophilic carbon of the isothiocyanate group. Then, a thiourea bond is formed between FITC and the protein without leaving groups.

The present experiment deals with a protein (bovine serum albumin, BSA) modification using a fluorescent probe, i.e. fluorescein isothiocyanate (FITC), and the subsequent determination of the macromolecule modification determined using absorbance spectroscopy. During this experiment students are asked to evaluate the molar extinction coefficient (ε) of the protein (BSA) and the fluorophore (FITC), bioconjugate BSA with FITC and learn how to purify and characterize the obtained conjugate. Students are thus allowed to learn different topics (modification of reactive groups of proteins, preparation, purification, isolation and characterization of conjugates) and use several laboratory techniques (pipetting, gel filtration, spectroscopy).

For its interdisciplinary character, this laboratory experiment is well-suited for students in third year of Bachelor degree in chemistry, biochemistry, biology and biotechnology and the time required for completing the described experiment is no longer than three lab sessions, each one taking about four hours. Students usually work in small groups (ideally two or up to four students each group) depending on the availability of working space and instruments. The accuracy of students’ results and response to the experiments were generally good (within 5-10% error).

留下评论