title: Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein.
abstract: Vitamin D3and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3and three metabolites: 1,25-dihydroxyvitamin D3[1,25(OH)2D3], 25-hydroxyvitamin D3(25OHD3) and 24,25-dihydroxyvitamin D3[24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3and 24,25(OH)2D3had higher affinity (0.3 μM) than 1,25(OH)2D3(5 μM), with the higher affinity of 25OHD3and 24,25(OH)2D3due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3(NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a KDof 50 μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2(Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2(Ph-491), structurally similar, had affinities of 180 and 170 μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3(MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3(Oxan-429) had affinities of 310 and 100 μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes.
authors.affiliation: Centro de Investigaciones Biomédicas (CINBIO), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain. pcanoa@uvigo.es.|Departamento de Química Orgánica, Facultad de Química and Instituto de Investigación Biomédica (IBI), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain. pcanoa@uvigo.es.
authors.forename: Pilar
authors.initials: P
authors.lastname: Canoa
authors.affiliation: Departamento de Química Orgánica, Facultad de Química and Instituto de Investigación Biomédica (IBI), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain.
authors.forename: Marcos L
authors.initials: ML
authors.lastname: Rivadulla
authors.affiliation: Bio-Rad Laboratories Ltd, Bio-Rad House, Maxted Road, Hemel Hempstead, Hertfordshire, HP2 7DX, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Popplewell
authors.affiliation: , Dishman, Nieuweweg 2a, 3901 BE, Veenendaal, The Netherlands.
authors.forename: René
authors.initials: R
authors.lastname: van Oosten
authors.affiliation: Departamento de Química Orgánica, Facultad de Química and Instituto de Investigación Biomédica (IBI), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain.
authors.forename: Generosa
authors.initials: G
authors.lastname: Gómez
authors.affiliation: Departamento de Química Orgánica, Facultad de Química and Instituto de Investigación Biomédica (IBI), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain.
authors.forename: Yagamare
authors.initials: Y
authors.lastname: Fall
mesh_terms: D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007700:Kinetics; D020349:Surface Plasmon Resonance / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism*; D014809:Vitamin D-Binding Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014809:Vitamin D-Binding Protein; D014807:Vitamin D
keywords: Affinity; Kinetics; Surface plasmon resonance; Vitamin D analogues; Vitamin D binding protein; Vitamin D metabolites