tailieunhanh - Báo cáo khoa học: Application of a fluorescent cobalamin analogue for analysis of the binding kinetics A study employing recombinant human transcobalamin and intrinsic factor

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 attach-ment facilitated detailed kinetic analysis of Cbl binding. | ễFEBS Journal Application of a fluorescent cobalamin analogue for analysis of the binding kinetics A study employing recombinant human transcobalamin and intrinsic factor Sergey N. Fedosov1 Charles B. Grissom2 Natalya U. Fedosova3 S0ren K. Moestrup4 Ebba Nex05 and Torben E. Petersen1 1 Protein Chemistry Laboratory Department of Molecular Biology University of Aarhus Denmark 2 Department of Chemistry University of Utah Salt Lake City UT USA 3 Department of Physiology and Biophysics University of Aarhus Denmark 4 Department of MedicalBiochemistry University of Aarhus Denmark 5 Department of ClinicalBiochemistry AS Aarhus University Hospital Denmark Keywords cobalamin fluorescence intrinsic factor transcobalamin Correspondence S. N. Fedosov Protein Chemistry Laboratory Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej10 8000 Aarhus C Denmark Fax 45 86 13 65 97 Tel 45 89 42 50 92 E-mail snf@ Received 9 June 2006 revised 31 July 2006 accepted 18 August 2006 doi 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 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 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 domaindomain interactions are the most important