Determination of substrates using poly(ethylene glycol)-stabilized dehydrogenase enzymes by microlitre per minute flow injection

Abstract

Flow injection (FI), at a flow rate of µl min^–1, is an effective method for enzymic substrate determinations using low concentrations of poly(ethylene glycol)(PEG)-stabilized soluble enzymes. PEG stabilizes dehydrogenase enzymes for at least several days by promoting sub-unit association. Band broadening of knitted open tubular reactors is reduced as flow rate decreases below 300 µl min^–1 and a small tubing diameter is important for a faster rate of absorbance signal increase with residence time. Small (0.5 µl) sample injections also ensure narrow FI peaks. The determination of several substrates such as pyruvate, lactate, and cortisone using appropriate PEG-stabilized enzymes is demonstrated with this FI instrument at 25 or 50 µl min^–1 with sample throughputs of the order of 2–3 min per sample. The determination of lactate in serum samples is also possible. The advantage of this method, sample throughput, is not sacrificed but enzyme consumption is considerably less, compared to standard ml min^–1 FI.