Preparation and characterization of bovine albumin isoforms.
Albumin undergoes changes in conformation and isomerizations by disulfide interchange of unknown biological significance. The aim of this study was to prepare and characterize albumin isoforms, which were stable under near physiological conditions. Modified albumins were obtained by urea denaturation and renaturation, and by aging at low ionic strength and alkaline pH in the presence of cysteine. We describe a cathodic electrophoresis technique, which allows the separation of albumin isoforms with greater positive charge. Differences between native and modified albumins were analyzed by new criteria based on the reactivity of the thiol and histidyl residues and on the susceptibility of the disulfide bonds to sulfitolysis. Modified albumins had, (i). a more cationic component which disappears by sulfitolysis of the disulfide bonds or by incubation with a glutathione redox system; (ii). higher reactivities of the free thiol group and of the histidyl residues, and; (iii). decreased fluorescence. These differences were not observed when processes were carried out on albumin with the thiol group blocked by iodacetic acid, but reappeared with the addition of cysteine. Renatured and aged albumins differed in the nature of the cationic component. Generation of albumin isoforms is dependent on the presence of a free thiol group and seems to involve thiol disulfide interchanges.
Gabaldón
María
M
Unidad de Histoquimia, Centro de Investigación, Hospital La Fe, Avenida Campanar 21, 46009, Valencia, Spain. gabaldon_mar@gva.es
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D007202:Indicators and Reagents; D010767:Phosphorylcholine / Q000031:analogs & derivatives*; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D020673:Protein Renaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014508:Urea
D004220:Disulfides; D007202:Indicators and Reagents; C437127:O,O-diethylphosphorylcholine iodide; D020033:Protein Isoforms; D013438:Sulfhydryl Compounds; D010767:Phosphorylcholine; D012710:Serum Albumin, Bovine; D006639:Histidine; D014508:Urea