Interference by sucrose in the chemical determination of citrulline with diacetyl monoxime

The chemical determination of citrulline with diacetyl monoxime is subject to interference by sucrose and fructose. Glucose, sorbitol, and mannitol do not interfere. Likewise, the presence of EDTA, 2-mercaptoethanol, and HEPES (N-2-hydroxyethylpiperazine-N′-2-ethane sulphonic acid) at concentrations normally employed in mitochondrial isolation media does not affect the assay. The use of this method to estimate citrulline formation by mitochondria prepared in sucrose containing medium may thus be subject to error unless the effect of sucrose is taken into account. For such studies of mitochondrial citrulline formation employing diacetyl monoxime as the method of citrulline assay, the use of a mannitol-based isolation medium is recommended.     

A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline

l-Citrulline constitutes a product of a number of enzymatic reactions. In the past a number of colorimetric methods for the determination of l-citrulline, upon its chemical modification with diacetyl monoxime at 95 degrees C, have been reported. However, all these methods are time- and material-consuming. In this work, using the same chemical reaction, a new method for the use in 96-well polystyrene microtiter plates was developed. The method is fast and requires substantially less material as the enzymatic reaction is performed in a volume of 60 microl. The applicability of this enzymatic assay was established using l-N(omega), N(omega)-dimethylarginine dimethylaminohydrolase, which generates l-citrulline from side-chain methylated derivatives of l-arginine. The detection limit for l-citrulline is about 0.2 nmol. In addition, our studies show that most commonly used biochemical buffers and buffer additives do not affect the assay. This method may prove useful in the studies of other l-citrulline producing enzymes including nitric oxide synthase.     

Effects of mechanical uncouplers, diacetyl monoxime, and cytochalasin-D on the electrophysiology of perfused mouse hearts

Chemical uncouplers diacetyl monoxime (DAM) and cytochalasin D (cyto-D) are used to abolish cardiac contractions in optical studies, yet alter intracellular Ca(2+) concentration ([Ca(2+)](i)) handling and vulnerability to arrhythmias in a species-dependent manner. The effects of uncouplers were investigated in perfused mouse hearts labeled with rhod-2/AM or 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) to map [Ca(2+)](i) transients (emission wavelength = 585 +/- 20 nm) and action potentials (APs) (emission wavelength > 610 nm; excitation wavelength = 530 +/- 20 nm). Confocal images showed that rhod-2 is primarily in the cytosol. DAM (15 mM) and cyto-D (5 microM) increased AP durations (APD(75) = 20.0 +/- 3 to 46.6 +/- 5 ms and 39.9 +/- 8 ms, respectively, n = 4) and refractory periods (45.14 +/- 12.1 to 82.5 +/- 3.5 ms and 78 +/- 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min and DAM by 10% gradually in 1 h (n = 5 each). Uncouplers did not alter the direction and gradient of repolarization, which progressed from apex to base in 15 +/- 3 ms. Peak systolic [Ca(2+)](i) increased with cyto-D from 743 +/- 47 (n = 8) to 944 +/- 17 nM (n = 3, P = 0.01) but decreased with DAM to 398 +/- 44 nM (n = 3, P < 0.01). Diastolic [Ca(2+)](i) was higher with cyto-D (544 +/- 80 nM, n = 3) and lower with DAM (224 +/- 31, n = 3) compared with controls (257 +/- 30 nM, n = 3). DAM prolonged [Ca(2+)](i) transients at 75% recovery (54.3 +/- 5 to 83.6 +/- 1.9 ms), whereas cyto-D had no effect (58.6 +/- 1.2 ms; n = 3). Burst pacing routinely elicited long-lasting ventricular tachycardia but not fibrillation. Uncouplers flattened the slope of AP restitution kinetic curves and blocked ventricular tachycardia induced by burst pacing.     

Estimation of nitrogenase using a colorimetric determination for ethylene

Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene.     

The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

The spectrophotometric determination of diacetyl

A new assay for the determination of diacetyl based on the colorimetric reaction with creatine and alpha-naphthol in an alkaline medium was developed. After a determination of optimal concentrations of the necessary reagents, the assay could easily detect as little as 0.5 micrograms of diacetyl. Since mammalian tissue extracts inhibit the reaction, distillation of the tissue filtrates before the assay was required. Rat heart, kidney, and liver were shown to contain diacetyl while brain was negative.     

Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles

Whether or not the excitation-contraction (E-C) uncoupler diacetyl monoxime (DAM) and cytochalacin D (Cyto D) alter the ventricular fibrillation (VF) activation patterns is unclear. We recorded single cell action potentials and performed optical mapping in isolated perfused swine right ventricles (RV) at different concentrations of DAM and Cyto D. Increasing the concentration of DAM results in progressively shortened action potential duration (APD) measured to 90% repolarization, reduced the slope of the APD restitition curve, decreased Kolmogorov-Sinai entropy, and reduced the number of VF wave fronts. In all RVs, 15-20 mmol/l DAM converted VF to ventricular tachycardia (VT). The VF could be reinduced after the DAM was washed out. In comparison, Cyto D (10-40 micromol/l) has no effects on APD restitution curve or the dynamics of VF. The effects of DAM on VF are associated with a reduced number of wave fronts and dynamic complexities in VF. These results are compatible with the restitution hypothesis of VF and suggest that DAM may be unsuitable as an E-C uncoupler for optical mapping studies of VF in the swine RVs.     

A colorimetric micro-method for the determination of glutathione

1. A rapid colorimetric and apparently specific micromethod for the determination of total glutathione in small amounts of tissue is described. Generally, less than 30mg. of tissue is sufficient and this is homogenized in ice-cold 3% metaphosphoric acid. The product is filtered through sintered glass and neutralized or diluted before being added to a cuvette containing phosphate buffer, pH7·1, 5,5′-dithiobis-(2-nitrobenzoic acid), EDTA and glutathione reductase. Addition of NADPH₂ to the system initiates a progressive reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) by catalytic amounts of GSH, and this causes a colour increase at 412mμ. The rate of this change, calculated over 5min., is proportional to the total amount of glutathione present, and consequently unknown concentrations may be determined by reference to standards. 2. A preparation (based on that of Racker, 1955) of a suitable sample of glutathione reductase from yeast is described. 3. A less specific and less sensitive determination of extracted thiol groups with 5,5′-dithiobis-(2-nitrobenzoic acid) at pH8·0, based on observations of Ellman (1959) and Jocelyn (1962), is also described. 4. Although the precise nature of the reaction is not known, evidence is put forward to support a process of cyclo-reduction. GSSG is reduced enzymically to GSH, which reacts with 5,5′-dithiobis-(2-nitrobenzoic acid) to produce a coloured ion: [Formula: see text] (E_max. 412mμ) and a mixed disulphide. This disulphide reacts with further quantities of GSH to liberate another ion and GSSG, which then re-enters the cycle.     

菖蒲过氧化物酶测定条件的研究

过氧化物酶是植物在低温等逆境条件下酶促防御系统的关键酶之一,其活性可以作为植物抗寒性的一个指标。以菖蒲为材料比较了反应体系、H2O2浓度、pH值及反应时间对过氧化物酶活性测定的影响。结果表明:过氧化物活性测定的较佳反应体系是3 mL;最佳H2O2浓度为0.1%;最佳磷酸缓冲液pH值为6.0;最佳反应时间为4min。该研究结果为人工湿地污水处理系统冬季植物的筛选与评价研究提供参考,对植物生理实验教学和相关科学研究有一定的参考价值。     

A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase

A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.