ABSTRACT
Examining the effects of different cosolutes on in vitro enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, in vivo milieu. Viewing cosolute in vitro influences on a model enzyme, calf intestinal alkaline phosphatase, as a combination of competitive and uncompetitive behaviors provided quantitative insights into their effects on catalysis. Observed decreases in the apparent specificity constant, K asp, caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme-substrate complex formation. This competitive inhibition appeared to be driven by osmotic stress. Dextran 6 K and sucrose strongly impeded the subsequent conversion of the bound substrate into a free product, which was marked by sharp reductions in V max, uncompetitive inhibition. For the same step, smaller noncarbohydrate cosolutes, triethylene glycol, polyethylene glycol 400, and betaine, also behaved as uncompetitive inhibitors but to a lesser extent. However, polyethylene glycol 8000 and 20,000 were uncompetitive activators, increasing V max. Polyethylene glycol of molecular weight 1000 displayed intermediate effects between these two groups of noncarbohydrate cosolutes. These results suggested that crowding has a strong influence on free product formation. The combination of competitive and uncompetitive effects and mixed behaviors, caused by the cosolutes on calf intestinal alkaline phosphatase kinetics, was consistent with the trends seen in similar enzyme-cosolute studies. It is proposed that the double-displacement mechanism of alkaline phosphatases, shared by many other enzymes, could be the root of this general observation.
ABSTRACT
Glycidyl methacrylate-ethylene dimethacrylate (GMA-co-EDMA) based monolithic porous layer open tubular (monoPLOT) columns (0.05 mm I.D., monolithic layer thickness ≈ 5 µm) have been fabricated using an automated column scanning technique, providing UV polymerisation at 365 nm. Columns were chemically modified to obtain desired diol groups on the surface, and the longitudinal homogeneity of the stationary phase was profiled using scanning capacitively coupled contactless conductivity detector (sC4D), before and after such modification. Using the automated scanning polymerisation technique, column-to-column production reproducibility, including longitudinal phase thickness, was within ±5% RSD. The prepared columns were tested to evaluate their liquid chromatographic stationary phase selectivity, efficiency and reproducibility, with a series of test protein mixtures. Under optimised gradient conditions, the separation of up to 8 proteins was demonstrated on the open tubular column (510 × 0.05 mm I.D.), with a column pressure drop of <1.5 MPa.
ABSTRACT
Evanescent wave-initiated photopolymerisation in an optically wave guiding PTFE-coated fused silica capillary using light-emitting diode as a light source, is established here as a way to fabricate monolithic porous layer open-tubular capillary columns with a potential in capillary separation methods; application of the obtained open-tubular columns as enzymatic microreactors for on-line protein digestion is demonstrated.
