title: Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.
abstract: Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.
authors.affiliation: Department of Chemistry, University of Modena, via Campi 183, 41100 Modena, Italy
authors.forename: M
authors.initials: M
authors.lastname: Borsari
authors.forename: E
authors.initials: E
authors.lastname: Dikaya
authors.forename: A
authors.initials: A
authors.lastname: Dikiy
authors.forename: M V
authors.initials: MV
authors.lastname: Gonchar
authors.forename: M M
authors.initials: MM
authors.lastname: Maidan
authors.forename: R
authors.initials: R
authors.lastname: Pierattelli
authors.forename: A A
authors.initials: AA
authors.lastname: Sibirny
mesh_terms: D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group