Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase Satoshi Igarashi a , Koji Sode b, * a Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki, 305-0044, Japan b Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan Received 25 December 2003; accepted 18 June 2004 Abstract In order to study in greater detail the subunit interaction of the homodimeric soluble quinoprotein glucose dehydrogenase (PQQGDH-B), we developed an effective method of creating heterodimeric PQQGDH-B. Two different homodimers are combined, one of which has a polyarginine tail (Arg-tail), and subjected to a protein dissociation/redimerization procedure. Separation of the mixture by cation exchange chromatography results in three peaks showing GDH activity, eluting at 133, 231 and 273 mM NaCl concentration. These peaks were determined to correspond to the Arg-tailless homodimer, heterodimer, and Arg-tailed homodimer, respectively. To test this approach, we constructed and characterized heterodimeric PQQGDH-B composed of native (wild-type) and inactive mutant (His168Gln) subunits. The heterodimeric wild-type-His168Gln showed slightly decreased GDH activity and almost identical substrate specificity profile to the wild-type enzyme. Moreover, the Hill coefficient of the heterodimer was calculated as 1.13, indicating positive cooperativity. D 2004 Elsevier B.V. All rights reserved. Keywords: Pyrroloquinoline quinone (PQQ); Glucose dehydrogenase (GDH); Heterodimer; Cooperativity 1. Introduction Soluble quinoprotein glucose dehydrogenase (PQQGDH-B), because of its high catalytic efficiency (k cat /K m ) and independence of dissolved oxygen, is a promising 0165-022X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2004.06.009 * Corresponding author. Tel.: +81 42 388 7027; fax: +81 42 388 7027. E-mail address: sode@cc.tuat.ac.jp (K. Sode). J. Biochem. Biophys. Methods 61 (2004) 331 – 338 www.elsevier.com/locate/jbbm