L-cysteine assay

Analysis of Crude CMC by Manual and Automated L-Cysteine Assays... 

Table 1. Analysis of crude CMC by direct manual L-cysteine assay. 

Table 3. Analysis of CMC in liquid detergent formulations using direct manual and chromatography automated L-cysteine assay. 

Eskinja, M., Lamprecht, G., Scherer, G., and Schmid, E. R., Assay of S-ethyl-N-acetyl-L-cysteine in urine by high-performance liquid chromatography using post-column reaction detection, /. Chromatogr. B, 704, 159, 1997. 

The incubation mixture for assay of y-glutamylcysteine synthetase contained in a final volume of 0.3 mL 0.1 M Tris-HCl (pH 8.2), 6 mM ATP, 50 mM KC1, 6 mW dithiothreitol, 20 mM MgCl2, 3 mW L-cysteine, and 15 mM L-glutamic acid. The mixture was incubated at 37°C for 15 minutes to ensure the complete reduction of cysteine. The reaction was initiated by addition of hemolysate. Samples of 20 /xL were withdrawn at various times between 10 and 30 minutes and added to 50 /xL of 50 mM N-ethylmorpholine (pH 8.4) and 10 /xL of monobromobimane in acetonitrile. After incubation for 15 minutes in the dark at room temperature, the reaction was stopped by addition of 80 /xL of 10% sulfosalicylic acid. The volume was made up to 500/xL with water before centrifugation and analysis of the resulting supemate by HPLC. 

Elevated concentrations of plasma homocysteine (HCY) are related to an increased risk of cardiovascular disease, which exists in numerous forms in plasma, with the main form existing as a disulfide with itself, cysteine, or albumin. Therefore, the first step in the measurement involves treatment with a reducing agent, in this case dithiothreitol (DTT), to obtain HCY in its free form (Eq. 16.34). Some amino acids (e.g., L-cysteine and L-methionine) are present in human plasma at higher molar concentrations than HCY and may interfere with this assay. To avoid this possible interference, the highly selective enzymatic conversion of HCY to S-adenosyl-L-homocysteine (SAH), as shown in Eq. 16.34, is used. Both reactions (reduction and conjugation) are accomplished in 30 min at 34 °C. 

Cross-reactivity was study for similar molecules expected to be present in plasma. L-Cysteine (hydrochloride) and L-methionine at 5 and 4.5 mmol/L, respectively, were assayed in buffer. The observed polarization values correspond to HCY concentrations of 0.0 and 0.1 pmol/L, respectively. Thus, the antibody-binding reaction is sufficiently selective in the presence of these potential interferents. 

To evaluate the amount of acetaminophen bound to proteins, utilizing the competitive A-B ELISA or PCFIA, inhibition by unknown samples was compared with an assay standard prepared by derivatizing protein with NAPQI. For some experiments, 3-(N-acetyl-L-cystein-S-yDacetaminophen was used as an assay standard, in which case, the values obtained were corrected for differences in the relative inhibitory potency of 3-(N-acetyl-L-cystein- yl)acetaminophen and 3-Cys-A protein adduct (120 fmol/well and 2300 finol/well, respectively) (14). After dialysis, unknown samples were diluted to a final concentration of approximately 4 / g protein/assay well, assayed in duplicate, and expressed as nmoles of 3-Cys-A per mg of protein. The ELISA and PCFIA were shown to have similar limits of detection (20 pmole/mg protein) and to recognize the same itope as demonstrated by similar relative inhibitory potencies for N-acetylcysteine-acetaminophen, acetaminophen-bound... 

In a cultured human renal proximal tubular cell line, HK-2, a concentration-related increase in the level of ROS compared with controls was observed after 6 h exposure to ochratoxin A at 50-600 pmol/l. Pretreatment with A/-acetyl-L-cysteine, an antioxidant ROS scavenger, decreased the level of ROS and increased cell survival. A/-Acetyl-L-cysteine also reduced DNA damage at 50-200 pmol/l, as measured by the comet assay, but not at higher ochratoxin A concentrations of >400 pmol/l, at which the generation of ROS outpaced the reduction by A/-acetyl-L-cysteine (Arbillaga et al., 2007a). 

Low pressure column chromatography is characterized by the use of compressible column packings 025 urn) which require the use of low pumping pressures, e.g. hydrostatic pressure or peristaltic pumps, and extended analysis times in the region of 2-18 hours. Detection is frequently performed using automated assay systems, e.g. an L-cysteine sulphuric acid system, or by manual assay following collection of eluant fractions (1). 

Carboxymethyl cellulose (1 mg) - samples A, B, and C - was dissolved in distilled water (1 mL). Aliquots of 20 pi were added to 180 pL of distilled water to give aqueous solutions with final concentrations of 20pg/200pL. The L-cysteine-sulfuric acid (86%) reagent (1 mL) was added to these solutions, the mixture stirred and heated at 95°C for 3 min, cooled and the absorbencies measured spectrophotometrically at 415 nm. An aliquot of 100 pL was loaded on to the chromatographic column packed with Biogel P6 which was connected to an automated L-cysteine sulfuric acid assay. The same procedure was performed for these samples after 24 hours dialysis in order to remove the impurities present in the preparations. 

The chemical analysis used for measuring the total sugar content of crude CMC samples by manual L cysteine sulfuric acid assay was affected by the impurities present in the preparations since higher percentages of glucose were obtained when compared to the stated values of approximately 30%. However, after dialysis of the preparations, these percentages came down to the expected values (Table 1). 

The percentages of glucose produced from crude CMC samples were the same before and after dialysis when assayed by an automated L-cysteine-sulfuric acid system which was connected to a chromagraphic column packed with BiogelR P6. This is due to the capacity of the chromatographic system to remove the impurities present in the CMC samples (Table 2). The... 

Aliquots (75 pi of 1% solution) of the supernatants of the enzymic hydrolysates of starches were injected into a water-jacketed column (50 x 1.0 cm i.d.) packed with Biogel P2 (400 mesh particle size) and maintained at 60 C. The mobile phase was 0.1 M sodium chloride with a flow rate of 0.16 mL/min. The column eluent was continuously monitored using an automated L-cysteine sulfuric acid assay. The system was standardized by injecting a mixture of glucose and malto-oligosaccharides of known composition. 

In addition, the enzyme was converted to an inactive form during purification procedure, and the inactive enzyme was activated by preincubation with L-cysteine under anaerobic condition. (2) The activated enzyme was rapidly and irreversibly inactivated during aerobic assay condition and this inactivation was prevented by a distinct cytoplasmic protein in rat liver, called protein-A. On the basis of these findings, the enzyme from cytosol of rat liver was purified to homogeneity by Yamaguchi et al.l. In the present report is discussed the significance and the regulatory role of cysteine... 

Effects of temperature and of the concentration of L-cysteine on the anaerobic activation of cysteine dioxygenase. A, Cysteine dixoygenase was preincubated with 10 mM L-cysteine under anaerobic condition at 10, 20, 30 and 37 , and the volume of preincubation mixture was 0.05 ml. B, Cysteine dioxygenase was preincubated under normal activation conditions except the concentration of L-cysteine given in the figure, and the volume of preincubation mixture was 0.05 ml. After mixture volume was adjusted to 1.0 ml, the enzyme activity was assayed under standard assay con-citions. (From Yamaguchi et al., ref. 13, with permission)... 

The true initial velocity(Vo) of the reaction can also be calculated by extrapolation of the plots of log(Vt) versus time back to zero time. Using Vo values, the apparent Km for L-cysteine in the absence of protein-A was obtained to be 6.7 x 10 M. Since this Km value did not significantly differ from that in the presence of protein-A, protein-A did not affect the affinity of enzyme for cysteine. Since the irreversible inactivation during assay occurred in the... 

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