CAPILLARY ZONE ELECTROPHORETIC RESOLUTION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 GLYCOFORMS - AN INVESTIGATION INTO THE SEPARATION MECHANISMS FOR AN EXQUISITE SEPARATION

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is a disulfid e-linked homodimeric glycoprotein (M(r) = 30 000) which induces bone f ormation in vivo in several animal model systems. In this paper, we re port the separation of a homogeneous rhBMP-2 sample into nine peaks by capillary zone electrophoresis (CZE), using a simple, pH 2.5, phospha te buffer containing no additives. The nine peaks have been identified to be glycoforms of rhBMP-2 [designated as (rhBMP-2)(2)-(GlcNAc)(4)(M an(Z)), where Z varies from 10 to 18]. The difference between any adja cent pair of peaks is only one mannose residue (M(r) = 162). The abili ty of CZE to resolve rhBMP-2 glycoforms having the same charge and dif fering only 0.5% in molecular mass, without resorting to chemical comp lexation, is both unexpected and intriguing. Possible mechanisms expla ining how the additional mannose can affect the mobility of rhBMP-2 gl ycoforms were explored. Zeta potentials of various glycoforms were cal culated from their mobilities and interpreted in light of diffuse doub le layer parameters. Our results suggest that CZE employing a low-pH b uffer, where proteins are highly charged, may be uniquely suitable for complex protein glycoform analysis.