Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.
The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.
Environmental and Molecular Sciences Laboratory, Richland, WA 99352, USA.
S
S
Martinović
Pasa-Tolíc
C
C
Masselon
P K
PK
Jensen
C L
CL
Stone
R D
RD
Smith
D000426:Alcohol Dehydrogenase / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007527:Isoenzymes / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis
D007527:Isoenzymes; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase