Microplate screening assay for binding of ligands to bovine or reindeer beta-lactoglobulins.
Several analytical methods have been used to determine whether ligands bind to bovine beta-lactoglobulin (betaLG). The most common methods are based on fluorescence quenching. We have miniaturised this method from a quartz cell to a 96-well plate. The miniaturisation was evaluated using retinol. The binding constants between the two methods demonstrated a good correlation. The 96-well plate method is much faster and allows many references to be used in the same analysis. The miniaturised method was used to study the binding of three different ligands (4-HPR, arotinoid, warfarinyl palmitate) modelled to bind to betaLG. The binding data showed that all of these ligands bound to betaLG. The method was further used to demonstrate that reindeer betaLG could also bind the four ligands in the same way as bovine betaLG. Because one aim is to use bovine and reindeer betaLG as a binder molecule for aliments in e.g. functional food or for drugs, the influence of pH was also studied and demonstrated that short-term acidic conditions had only a slight effect on the binding properties.
Riihimäki
Laura
L
Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
Aitio
Olli
O
Vahermo
Mikko
M
Heikura
Jonna
J
Valkonen
Kaija
K
Virtanen
Vesa
V
Yli-Kauhaluoma
Jari
J
Vuorela
Pia
P
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002417:Cattle; D017313:Fenretinide / Q000737:chemistry / Q000378:metabolism; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D012053:Reindeer; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity
D007782:Lactoglobulins; D008024:Ligands; D012176:Retinoids; D017313:Fenretinide