Application of a fluorescent cobalamin analogue for analysis of the binding kinetics. A study employing recombinant human transcobalamin and intrinsic factor

Sergey N Fedosov, Charles B Grissom, Natalya U Fedosova, Søren K Moestrup, Ebba Nexø, Torben E Petersen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstrakt

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 (K(d) = 5 x 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 + IF(30) and CBC + IF(20) when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF(30)-CBC-IF(20) with higher fluorescent signal. However, dissociation of IF(30)-CBC-IF(20) 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 K(d) = 1.5 x 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.

OriginalsprogEngelsk
TidsskriftF E B S Journal
Vol/bind273
Udgave nummer20
Sider (fra-til)4742-53
Antal sider12
ISSN1742-464X
DOI
StatusUdgivet - 2006

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