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Rationally Designed Ligands for Use in Affinity Chromatography 97
(1), 1,5-diaminopentane (2), tyramine (3), m-xylylenediamine (4), phenethylamine
(5), isoamylamine (6), 4-aminobutyric acid (7), 4-aminobenzamide (8), 1-aminopropan-2-ol
(9), -alanine (10), 2-methylbutylamine (11), 4-aminobutyramide
(12), whereas ammonia was considered as a control (0). Apart from compound
12 (synthesized according to procedure described by Boeijen and Liskamp (11)),
all the amines were commercially available, and hazards and toxicity data were
considered individually for each compound according to suppliers’ recommendations.
The compounds must be dissolved in an appropriate buffer, either an aqueous
solution (usually for hydrophilic amines) or an organic solvent such as a 50%
(v/v) aqueous solution in dimethylformamide (DMF). In any case, usually 1 molar
equivalent of NaHCO 3 is added in order to neutralize the HCl released during
nucleophilic substitution. Caution: DMF is harmful and considered a potential
carcinogen. Should be handled in a fume hood.
2.3. Assessing the Affinity of Ligands for the Target Protein
1. Proteins tested: The human proteins utilized in the search of a PpL mimetic ligand
are widely available from various suppliers and included IgG, Fab, F(ab´) 2 and
Fc (crystallizable fragment). Reagent grade proteins with ≥95% purity must be
used. Caution: Human proteins are considered biohazardous, handle as if capable
of transmitting infectious agents.
2. Buffers: The buffers used for the screening of the ligands vary from case to case,
being dependent on the type of protein studied, the standard conditions recommended
for its use and the type of interactions exploited in the affinity purification.
The regeneration buffer usually utilized is 0.1 M NaOH in 30% (v/v) isopropanol.
The regeneration buffer is used to remove any physically adsorbed ligand prior
to screening and after the screening procedure to remove retained protein. Special
care should be taken when using iso-propanol (Hazards: flammable, irritant to eyes,
respiratory system, and skin). Toxicity data: LD50 10g/kg oral, human (Should be
handled with gloves, safety glasses and avoid vapors). The equilibration/binding
and elution buffers were selected for according to the usual operational conditions
used in PpL affinity chromatographic assays (12). The former consisted
of phosphate-buffered saline (PBS) (10 mM sodium phosphate, 150 mM NaCl,
pH 7.4) and the latter contained 0.1 M glycine–HCl pH 2 (1 M Tris–HCl, pH 9,
was then added to the elution samples to neutralize the pH).
2.3.1. Screening Techniques
1. Fluorescein isothiocyanate (FITC)-based screening: The requirements are as
follows.
a. Target protein: Must be conjugated with FITC-isomer I (F), and the conjugation
occurs through free amino groups of proteins or peptides, forming a
stable thiourea bond. Conjugated proteins can be bought from most suppliers
of biochemical products, but the protein (P) can also be chemically modified in