The Enzymes of Honey: Examination by Ionexchange Chromatography, Gel Filtration, and Starch-Gel Electrophoresis

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Abstract
Crude dialysed enzyme concentrates from several bulk honeys, comb honeys, and a ‘honey’ produced by sugar-feeding of caged bees, have been examined by chromatography on DEAE-cellulose, gel filtration through Sephadex G-200 and starch-gel electrophoresis with a high-resolution enzyme assay procedure. Ion-exchange chromatography divided crude honey α-glucosidase preparations into 3–9 closely spaced components. A preparation containing no plant constituent, obtained by sugar-feeding caged bees, was relatively unstable. Its α-glucosidase showed only a single very sharp elution peak from DEAE-cellulose; the α-glucosidase elution pattern of a preparation from honey stored by a free-flying colony of bees resembled that from a sample of bulk honey. All honey α-glucosidase preparations examined showed single bands of activity by Sephadex G-200 gel filtration, with approximate molecular weights about 51 000. Molecular weight of the amylase is about 24 000; for honey glucose oxidase two fractions were obtained, corresponding to molecular weights about 120 000 and > 200 000. Starch-gel electrophoresis showed the α-glucosidase complex to have from 7 to a maximum of 18 components (isozymes), the latter in a bulk clover honey and the former in a single sample of comb honey. The 13 isozymes of the α-glucosidase of one honey sample had a constant ratio of activity upon sucrose and maltose, further confirming the α-glucosidase nature of honey ‘invertase’. The α-glucosidase complex from sugar-fed ‘honey’ was much less stable and had a lower migration rate on starch-gel electrophoresis than those from any of the five floral honeys examined, which also varied in apparent rate of migration; this, in conjunction with ion-exchange results, implies an interaction between the bee-introduced α-glucosidase and plant nectar protein components.