Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Abstract

Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

“…Upon binding to IF, the solvent accessibility area of Cbl is reduced to Ϸ19% compared with Ϸ7% in TC (23). This difference does not seem to influence the ability of Cbl to bind to these proteins, as shown by recent studies involving binding of fluorescent analogues and surface plasmon resonance, which show that the affinity of Cbl is similar for all three Cbl-binding proteins (25,26). However, the absence of a His coordination bond and wide binding site in IF might allow Cbl to move in and out of IF freely compared with TC.…”

Phenylethynylcobalamin: A Light‐Stable and Thermolysis‐Resistant Organometallic Vitamin B12 Derivative Prepared by Radical Synthesis

“…[10,25] As assumed on the basis of the known crystal structures of these two B 12 binding proteins, [7] the organometallic B 12 derivative 3 had high affinities for them, similar as vitamin B 12 (5): 3 attached with similar binding rates to IF (k on = 93 mL mol À1 s À1 ) and to TC (k on = 129 mL mol À1 s À1 ), and 3 also detached with a k off rate (1.5 10 À7 s À1 ), similar to 5 (Supporting Information, Figures S16 and S17). Therefore, 3 is likely to undergo uptake in humans and mammals with similar efficiency as vitamin B 12 and as other natural cobalamins.…”

Organometallic cobalamin anticancer derivatives for targeted prodrug delivery via transcobalamin-mediated uptake

“…1) was performed as previously established. 25 Indeed, since the CBC interaction with TCII induces an enhancement of its luminescence intensity, the binding of competitive Cbl derivatives can be directly monitored. The addition of TCII-recognized B 12 analogues quenches the luminescence of the TCII-CBC complex in proportion to the level of competition established.…”