The pH‐Stat and Its Use in Biochemistry

Jacobsen, C. F.; Léonis, J; Linderstrøm‐Lang, K.; Ottesen, Martin

doi:10.1002/9780470110201.ch5

Cited by 152 publications

(28 citation statements)

References 15 publications

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“…These include static procedures such as the titration of the acid formed in the reaction [5], the use of radiolabeled ACh [6] or colorimetric methods using Ellman's reagent [7]. The latter method is the most widely used to measure the activity of AChE and its decrease in the presence of inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Martı́n-Biosca

Asensi-Bernardi

Villanueva-Camañas

et al. 2009

J of Separation Science

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In this study the development of a procedure based on capillary electrophoresis after enzymatic reaction at capillary inlet methodology for the screening and in vitro evaluation of the biological activity of acetylcholinesterase (AChE) inhibitors is presented. The progress of the enzymatic reaction of the hydrolysis of acetylthiocholine at pH 8 in the presence of AChE and the inhibitor studied is determined by measuring at 230 nm the peak area of the reaction product thiocholine (TCh). In the method employed the capillary was first filled with 30 mM borate-phosphate buffer (pH 8.0) and subsequently, plugs of: (i) water, (ii) AChE solution, (iii) substrate solution with or without inhibitor, (iv) AChE solution, and (v) water, were hydrodynamically injected into the capillary, and were allowed to stand (and react) during a waiting period of 2 min. The applicability of the proposed methodology to estimate different kinetic parameters of interest such as inhibition constants K(i), identification of inhibitory action mechanism and IC(50), is evaluated using compounds with known activity, tacrine edrophonium, and neostigmine. The results obtained are compared with bibliographic values and confirm the effectiveness of the methodology proposed. Finally a method for AChE Inhibitor screening is proposed.

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Section: Introductionmentioning

confidence: 99%

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Martı́n-Biosca

Asensi-Bernardi

Villanueva-Camañas

et al. 2009

J of Separation Science

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“…The measuring principle of a titrimetric sensor (or a pH-STAT) relies on a pH controller to keep the pH in the reactor at a defined setpoint through addition of acid and/or base (Jacobsen et al, 1957). The hydrogen ion production (or consumption) rate (HPR) in the reactor caused by the biological and/or physicochemical processes is thus measured as the addition rate of hydroxyl (or hydrogen) ions.…”

Section: Introductionmentioning

confidence: 99%

Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off‐gas CO₂ measurements

Pratt

Yuan

Keller

2004

Biotech & Bioengineering

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A method for detailed investigation of aerobic carbon degradation processes by microorganisms is presented. The method relies on an integrated use of the respirometric, titrimetric, and off-gas CO(2) measurements. The oxygen uptake rate (OUR), hydrogen ion production rate (HPR), and the carbon dioxide transfer rate (CTR) resulting from the biological as well as physicochemical processes, coupled with a metabolic model characterizing both the growth and carbon storage processes, enables the comprehensive study of the carbon degradation processes. The method allows the formation of carbon storage products and the biomass growth rates to be estimated without requiring any off-line biomass or liquid-phase measurements, although the practical identifiability of the system could be improved with additional measurements. Furthermore, the combined yield for biomass growth and carbon storage is identifiable, along with the affinity constant with respect to the carbon substrate. However, the individual yields for growth and carbon storage are not identifiable without further knowledge about the metabolic pathways employed by the microorganisms in the carbon conversion. This is true even when more process variables are measured. The method is applied to the aerobic carbon substrate degradation by a full-scale sludge using acetate as an example carbon source. The sludge was able to quickly take up the substrate and store it as poly-beta-hydroxybutyrate (PHB). The PHB formation rate was a few times faster than the biomass growth rate, which was confirmed by off-line liquid- and solid-phase analysis. The estimated combined yield for biomass growth and carbon storage compared closely to that determined from the theoretical yields reported in literature based on thermodynamics. This suggests that the theoretical yields may be used as default parameters for modeling purposes.

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“…Papain activity in absence and presence of natural inhibitors I, II, and III Papain activity was determined according to Kirsh and Igelströn (1966) with N-carbobenzoxiglycil pnitrophenyl ester (Z-Gly-pNP) and also using α-Nbenzoyl-L-arginin ethyl ester (BAEE) as substracts (Jacobsen et al, 1957). The reactions were carried out in a thermostatic cuvette at 25º C. A Beckman model SS-2 potentiometer sensitive to variations of 0.01 units in pH was used and Tris was added with a Nimetrics microsyringe capable of measuring amounts up to 0.1 µL.…”

Section: Chromatography In Sp-sephadex C-25mentioning

confidence: 99%

Isolation of natural inhibitors of papain obtained from Carica papaya latex

Contiero

Goulart

2004

Braz. arch. biol. technol.

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Studies were carried out to natural papain inhibitor from papaya latex. Fresh latex from green fruits of Carica papaya was collected and immediately transported in ice bath to the lab, from which three fractions with inhibitor effect of esterase papain activity were isolated by latex dialysis, Sephadex G-25 gel filtration and ionic exchange chromatography in SP-Sephadex C-25. The isolated fractions, identified as inhibitors I and II, showed a negative reaction with ninhydrin; however, the fraction identified as P-III showed positive reaction with ninhydrin. Kine tics data showed non-competitive inhibition (inhibitor I) and uncompetitive (inhibitors II and P -III).

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The pH‐Stat and Its Use in Biochemistry

Cited by 152 publications

References 15 publications

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off‐gas CO₂ measurements

Isolation of natural inhibitors of papain obtained from Carica papaya latex

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The pH‐Stat and Its Use in Biochemistry

Cited by 152 publications

References 15 publications

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off‐gas CO2 measurements

Isolation of natural inhibitors of papain obtained from Carica papaya latex

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Product

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Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off‐gas CO₂ measurements