Measurement of ATP And Ligand-ATP Conjugates by Enzymic Cycling with Co-Immobilized Hexokinase and Pyruvate Kinase
Various methods have been devised for monitoring specific protein-binding assays with enzymic reactions (1–4). In one method the ligand is coupled covalently to NAD; and this conjugate is measured at low levels by means of enzymic cycling reactions (2). The limit of sensitivity of specific protein-binding assays is de-termined in part by the sensitivity of the enzymic cycling reactions. In principle, the sensitivity achievable with this method should be dependent on cycling rates and reaction times; however, it is often limited by the level of cofactor contaminating the enzymes which are used at extraordinarily high levels.
KeywordsImmobilize Enzyme Pyruvate Kinase Soluble Enzyme Cycling Rate Competitive Binding Assay
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