Research article

Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity

Fatemeh Bootorabi^1,2 , Janne Jänis³ , Jarkko Valjakka¹ , Sari Isoniemi³ , Pirjo Vainiotalo³ , Daniela Vullo⁴ , Claudiu T Supuran⁴ , Abdul Waheed⁵ , William S Sly⁵ , Onni Niemelä⁶ and Seppo Parkkila^1,2

¹Institute of Medical Technology, Tampere University Hospital, 33520 Tampere, Finland

²School of Medicine, University of Tampere and Tampere University Hospital, 33520 Tampere, Finland

³Department of Chemistry, University of Joensuu, 80101 Joensuu, Finland

⁴Università degli studi di Firenze, Laboratorio di Chimica Bioinorganica, I-50019 Sesto Fiorentino (Firenze), Italy

⁵Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, 63104 Missouri, USA

⁶Department of Laboratory Medicine and Medical Research Unit, Seinäjoki Central Hospital and University of Tampere, 60220 Seinäjoki, Finland

author email corresponding author email

BMC Biochemistry 2008, 9:32doi:10.1186/1471-2091-9-32

Published:	27 November 2008

Abstract

Background

Acetaldehyde, the first metabolite of ethanol, can generate covalent modifications of proteins and cellular constituents. However, functional consequences of such modification remain poorly defined. In the present study, we examined acetaldehyde reaction with human carbonic anhydrase (CA) isozyme II, which has several features that make it a suitable target protein: It is widely expressed, its enzymatic activity can be monitored, its structural and catalytic properties are known, and it contains 24 lysine residues, which are accessible sites for aldehyde reaction.

Results

Acetaldehyde treatment in the absence and presence of a reducing agent (NaBH₃(CN)) caused shifts in the pI values of CA II. SDS-PAGE indicated a shift toward a slightly higher molecular mass. High-resolution mass spectra of CA II, measured with and without NaBH₃(CN), indicated the presence of an unmodified protein, as expected. Mass spectra of CA II treated with acetaldehyde revealed a modified protein form (+26 Da), consistent with a "Schiff base" formation between acetaldehyde and one of the primary NH₂ groups (e.g., in lysine side chain) in the protein structure. This reaction was highly specific, given the relative abundance of over 90% of the modified protein. In reducing conditions, each CA II molecule had reacted with 9–19 (14 on average) acetaldehyde molecules (+28 Da), consistent with further reduction of the "Schiff bases" to substituted amines (N-ethyllysine residues). The acetaldehyde-modified protein showed decreased CA enzymatic activity.

Conclusion

The acetaldehyde-derived modifications in CA II molecule may have physiological consequences in alcoholic patients.