Human serum albumin bearing covalently attached iron(II) porphyrins as O2-coordination sites.
Tetrakis{(alpha,alpha,alpha,alpha-o-pivalamido)phenyl}porphinatoiron(II) with a bifunctional tail possessing an axially coordinated imidazolyl group and a protein attachable succinimidyl(glutamyl) group (FeP-GluSu) has been synthesized. It can efficiently react with the lysine residues of recombinant human serum albumin (rHSA), giving a new albumin-heme conjugate [rHSA(FeP-Glu)]. MALDI-TOFMS showed a distinct molecular ion peak at m/z 70 643, which indicates that three FeP-Glu molecules were covalently linked to the rHSA scaffold. The binding number of FeP-Glu is approximately three (mol/mol) and independent of the mixing ratio. The CD spectrum and Native PAGE revealed that the albumin structure remained unaltered after the covalent bonding of the hemes. This rHSA(FeP-Glu) conjugate can bind and release O2 reversibly under physiological conditions (pH 7.3, 37 degrees C) in the same manner as hemoglobin and myoglobin. The O2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O2-binding affinity [P(1/2)O2: 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.
Wang
Rong-Min
RM
Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Komatsu
Teruyuki
T
Nakagawa
Akito
A
Tsuchida
Eishun
E
D000818:Animals; D001665:Binding Sites; D018698:Glutamic Acid / Q000737:chemistry; D006420:Hemeproteins / Q000138:chemical synthesis* / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008652:Mesoporphyrins / Q000138:chemical synthesis / Q000378:metabolism*; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry
D006420:Hemeproteins; D007093:Imidazoles; D008652:Mesoporphyrins; D012709:Serum Albumin; D013388:Succinimides; C032620:succinimide; D018698:Glutamic Acid; C036382:iron (II) meso-tetra(alpha,alpha,alpha,alpha-o-pivalamidophenyl)porphyrin; C029899:imidazole; D008239:Lysine; D010100:Oxygen