Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.
The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.
Noy
N
N
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Slosberg
E
E
Scarlata
S
S
D001665:Binding Sites; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011228:Prealbumin / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
D011228:Prealbumin; D012177:Retinol-Binding Proteins; D014801:Vitamin A