Flow injection analysis for L-lactate with immobilized lactate dehydrogenase

Immobilized lactate dehydrogenase (LDH) is used for determination of L-lactate in a continuous flow system. The LDH is immobilized by reaction with glutaraldehyde onto the surface of alkylamino-bonded silica gel and packed into a column in the flow system. The reduction of NAD⁺ occurs simultaneously, and the NADH formed is detected amperometrically. The peak current is linearly related to the L-lactate concentration in the range 1–80 × 10^-6 M; 30 samples h^-1 can be analyzed with a r.s.d. of 0.5–1.5%. The immobilized LDH retains over 90% of its initial activity after repetitive use for 3 months.     

Determination of the complex stability of zinc with carbonic anhydrase in sea-water.

Carbonic anhydrase (CA) is inactive unless associated with zinc, with possible substitution by cobalt. In this work, the complexation of zinc by CA was determined in sea-water using cathodic stripping voltammetry (CSV) with ligand competition. The zinc was found to be released from the CA over a period of 3 h when equilibrated with a competing complexing ligand and the complex was re-formed with the CA when zinc was added. A value of 8.90+/-0.27 was found for logK'ZnCA where K'ZnCA is the conditional stability constant for the complex of Zn2+ with CA in pH 8 sea-water. A value for the molecular weight of CA was calculated from its equivalent ligand concentration (in nM) obtained by titrations with zinc at various CA concentrations (1-4 mg l(-1)). The value found (34740 g mol(-1)) for the molecular weight is consistent with values found previously by other methods (29000-31000 g mol(-1)) confirming that the stoichiometry of the complex between zinc and CA is 1:1. This work confirms that the zinc-CA complex is reversible and that the interaction between zinc and CA can be determined using CSV with ligand competition.     

Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors

Hydroxamates (R-CONHOH) have been scarcely investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). An inhibition/structural study of PhCONHOH is reported against all human isoforms. Comparing aliphatic (R = Me and CF(3)) and aromatic (R = Ph) hydroxamates as CAIs, we prove that CONHOH is a versatile zinc binding group. Depending on the nature of the R moiety, it can adopt different coordination modes to the catalytic ion within the CA active site.     

Flow injection analysis of trace hydrogen peroxide using an immobilized enzyme reactor

Summary Flow Injection Analysis of Trace Hydrogen Peroxide Using an Immobilized Enzyme Reactor Sub-part-per-billion levels of aqueous hydrogen peroxide have been determined with a flow injection analysis system employing a single bead string reactor composed of horseradish peroxidase covalently bound to an animated macroporous polymeric adsorbent with glutaraldehyde and a passive cation exchange membrane reactor to alter pH. The chemistry relies on the peroxidase mediated oxidation of nonfluorescentp-hydroxyphenylacetate to its fluorescent dimer. The advantage of the system includes rapid throughout rates (40 samples/h), excellent detection limits (0.3 ppb H₂O₂) and large dynamic range of linear response (1 ppb-1 ppm). However, the immobilized enzyme is not useful for the analysis of organic peroxides which act as inhibitors.     

Two-prong inhibitors for human carbonic anhydrase II

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.     

A comparative study of the catalytic mechanisms of the zinc and cadmium containing carbonic anhydrase

The catalytic mechanism for the conversion of carbon dioxide to hydrogen carbonate by a cadmium containing carbonic anhydrase was explored at density functional level employing two different models to simulate the active center of the enzyme. In the first model, the histidine residues around the metal ion were replaced with imidazole groups. Instead, in the second one, the simplest model was extended introducing two amino acidic residues generally present in the neighbor of enzyme and a deep water molecule. The results showed that cadmium carbonic anhydrase follows a reaction mechanism that is favored thermodynamically but not kinetically with respect to that of the most usual zinc-containing enzyme, both in a vacuum and in a protein environment.     

Flow injection determination of Pb and Cd traces with graphite furnace atomic absorption spectrometry

The preconcentration and recovery of lead and cadmium traces at ng l(-1) level were evaluated in standard solutions and natural aqueous samples using a FIAS (Flow Injection Atomic Spectrometry) apparatus. The method is based on retention of the complex formed between Pb or Cd and 1,2-dihydroxy-3,5-benzendisulphonic acid (Tiron) on a macroporous anion-exchange resin. The recovery of the analytes was obtained by elution with 0.1 M HCl and their determination was performed by Graphite Furnace Atomic Absorption Spectrometry (GFAAS). The detection limits were 9 and 7 ng l(-1) for Pb and Cd respectively. The effects of sample solution pH and composition and of interfering agents as well as reagent purity are discussed. The technique was applied to the analysis of natural waters.     

New computational evidence for the catalytic mechanism of carbonic anhydrase

Some aspects of the catalytic mechanism of HCA have been investigated. Either a zinc-bound water or a zinc-bound hydroxide has been considered as a nucleophile attacking CO ₂. No reaction path exists in the former case, while a transition state for the nucleophilic attack has been located in the latter (barrier of 7.6 kcal mol⁻¹). This activation energy is determined by the breaking of the hydrogen-bond network that shields the zinc-bound hydroxide when the CO ₂ molecule approaches the reaction center. No ambiguity exists about the mechanism for the internal rearrangement of the zinc–bicarbonate complex. The rotation pathway (Lindskog mechanism) proposed by many authors is too energy demanding since it causes the breaking of the hydrogen-bond network around the bicarbonate. The only possible rearrangement mechanism is a proton transfer (Lipscomb) that occurs in two steps (each step corresponding to a double proton transfer) and involves the Thr199 residue as a proton shuttle. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Contribution to the Fernando Bernardi Memorial Issue.     

Flow injection analysis for monitoring chemiluminescent reactions

Various coiled flow cells are tested for monitoring the chemiluminescence produced by the cobalt-catalysed oxidation of luminol by hydrogen peroxide and the fluorescein-sensitized oxidation of sulphide by sodium hypochlorite. When a 6-coil cell is used, 1O^?3–1OO ng of Co²⁺ and 1–1000 ng of S² can be determined in 10- and 100-μl samples, respectively.     

Flow injection analysis for glucose by the combined use of an immobilized glucose oxidase reactor and a peroxidase electrode

The amperometric peroxidase electrode measures hexacyanoferrate(III), produced by hydrogen peroxide, which is generated by injecting a 2μl sample into a reactor of immobilized glucose oxidase covalently bound to silica gel. The peak current is linearly related to the glucose concentration in the range 0.05–10 g l^?1; sample throughput is about 100 h^?1. Ascorbic acid (? 0.5 mM) does not interfere.     

Determination of cholesterol by flow injection analysis with immobilized cholesterol oxidase

The injected sample passes through a column of enzyme immobilized on controlled pore glass, at pH 7.0, and the hydrogen peroxide produced is detected amperometrically. As little as 0.2 μg of cholesterol can be detected. The method is applied to blood serum, wax-wool alcohol and an extract of butter.     

A caged hydrophobic inhibitor of carbonic anhydrase II

[formula: see text] A tight-binding, hydrophobic inhibitor of carbonic anhydrase II has been masked with a water-solubilizing, photolabile group derived from o-nitrophenylglycine. This caged inhibitor represents our first effort at the site-specific delivery of prodrugs that can be activated by light. Via this approach, we have begun to address the problems of water insolubility and systemic side effects on administration of tight-binding inhibitors of carbonic anhydrase.     

A new synthesis of difluoromethanesulfonamides--a novel pharmacophore for carbonic anhydrase inhibition

Preparation of the key intermediate carboxydifluoromethanesulfonamide provides direct synthetic access to a wide range of novel difluoromethanesulfonamides, including the acetazolamide analogue (2-ethanoylamino-1,3,4-thiadiazol-5-yl)-difluoromethanesulfonamide. Their water solubility and stability, ether partition coefficient, pK(a) and submicromolar dissociation constants for human carbonic anhydrase isozyme II (HCA II) make them promising candidates for topical glaucoma therapy.     

Flow injection determination of ethanol in whole blood using immobilized enzymes

Enzymatic methods for the determination of ethanol in whole blood are proposed. They use different types of detection and flow injection analysis (FIA) modes: fluorometric detection (use of normal FIA and stopped-flow/FIA); amperometric detection by monitoring of NADH (use of normal amperometric and pulse mode) and with the aid of a coupled enzymatic reaction (2,6-dichlorophenolindophenol/diaphorase). Determination ranges between 0.1 and 30.0 μg/ml are obtained (which in all cases comprise the legal range of ethanol in blood), with good precision and sampling frequency. The sensitivity of the methods can be manipulated by changing the injected sample volume or the pH.     

Flow injection in-valve-mini-column pretreatment combined with ion chromatography for cadmium, lead and zinc determination

A flow injection (FI) in-valve-mini-column packed with Chelex-100 resin is proposed for on-line sample pretreatment for some metal ions, namely, Cd(II), Pb(II) and Zn(II), prior to simultaneous determination using ion chromatography (IC). A solution containing a mixture of the cations was first passed through the in-valve-mini-column, followed by on-line elution. The eluate was then flowed further to an injection valve and was injected into an ion chromatograph. Conditions of the system were optimized. A single standard calibration was possible. The recoveries of cations were found to be in the range of 95–105%. The developed method was applied to the accurate analysis of zinc ore samples.     

Flow injection analysis with fourier transform infrared detection for clinical and process analysis

As the analysis of glucose and urea is of special interest in the field of clinical and process analysis, an enzymatic FIA-FTIR system is presented, based on the reactions of glucose to gluconic acid using glucose oxidase as well as of urea to ammonium carbonate catalyzed by urease. The method is tested with standard solutions, with samples simulating the blood composition and with a variety of fruit juices and soft drinks. Whereas the systematic error is within few percent, as concluded from the slope of the calibration graph, the reproducibility of the method is up to now not satisfactory for routine applications in process analysis and has to be improved.