Cysteine Solution Research Articles

Rapid Microdetermination of Hydroxyproline in Biomedical Samples by Flow Injection Analysis Using Cysteine as an Antioxidant

A new practical method for the rapid microdetermination of hydroxyproline (Hyp) in biomedical samples, such as tissue and urine, in the presence of a definite amount of cysteine as antioxidant was established by means of the modified KISO method using flow injection analysis. This method uses no organic solvent and requires only 9 min for each sample, which can be injected every 3 min (about 5 h is sufficient for 100 samples). A good linear relationship (r=0.99996) was obtained between the concentration of Hyp (0–80μg/ml) and the absorbance (0–0.514). In biomedical samples (hydrolyzates of rat stomach tissue), the recoveries (94.8–103.7%) and the reproducibility (the mean of RSD; 0.484% for ten kinds of rat tissues) were satisfactory. The correlation between values from this method and from the conventional KISO method was shown to be good enough (r=0.966) for hydrolyzate samples (N=95), when a proper amount of cysteine solution was added to both the sample and standard solutions and the concentration of the oxidant was optimized to Hyp contents.

A modified method for the determination of methylmercury by gas chromatography

A simple modification of the Westöö extraction procedure for methylmercury and its determination by gas chromatography (GC) is presented. The cysteine clean-up step has been modified, with use of cysteine-impregnated paper instead of cysteine solution. Methylmercury bromide is extracted from the sample into toluene and is selectively adsorbed on the cysteine paper. Interfering compounds are washed from the paper with toluene. The isolated methylmercury is set free with sulphuric acid containing bromide, extracted into benzene and determined by GC. The modification of the extraction procedure results in good recovery and reproducibility for various biological and environmental samples, good sensitivity with a detection limit of 0.1 ng/g, avoidance of difficulties arising from emulsion formation, cleaner chromatograms, and faster analysis. It is particularly suitable for determination of low levels of MeHg.

Conformation of ethyl (+)-(2S,3S)-3-{1-[N-(3-methylbutyl)amino]leucylcarbonyl}oxi-rane-2-carboxylate (loxistatin), a cysteine protease inhibitor: X-ray crystallographic and 1H nuclear magnetic resonance studies

In order to establish the conformational characteristics of a cysteine protease inhibitor, the crystal and solution conformations of loxistatin have been determined by X-ray crystallographic and 1H n.m.r. spectroscopic analyses. The molecules were arranged in the crystal as a series of infinite parallel β-sheet structures formed via intermolecular N-H ⋯ O hydrogen bonds. The loxistatin molecule has a flattened, curved conformation, which appears to be energetically stable. Similar conformations of loxistatin were also observed in (CD3)2SO and CDCl3 solutions. On the basis of the results obtained, the relation between the conformation of loxistatin and its inhibitory activity was discussed and compared with the stereostructure of the papain–substrate analogue complex.

けん気的に安定化した汚泥からの水銀の抽出

Mercury extraction was carried out using an anaerobically stabilized sludge originated from our laboratory wastewater treatment plant. Mercury in the sludge was not directly extracted not only with KI, KBr, Na2S2O3, ethylenediaminetetraacetate or cysteine solution at neutral pH, but also with 1.0 mol/l HCl. However, 20 per cent of mercury was extracted with 0.1 mol/l NaOH. On the other hand, 55 per cent of mercury was extracted with 0.1 mol/l KI at neutral pH when the anaerobic sludge was aerated for 6 days. Treatment of the aerated sludge with hydrogen peroxide solution, followed by treatment with KI solution, resulted in 85 per cent extraction of mercury. Most of mercury remaining in the sludge could be extracted by repeated treatment with KI solution. Mercury in the anaerobic sludge could also be extracted by aerating the sludge in 0.1 mol/l KI. Further more, when the anaerobic sludge was treated with Ca (OCl)2 at pH 8 and at pH lower than 2, 20 and 80 per cent of mercury were extracted. Other metal ions, such as Cu2+, Cd2+, Pb2+ and Zn2+, were scarcely extracted with 0.1 mol/l KI from the anaerobic sludge as well as aerobic or hydrogen peroxide treated sludges. From these results, it was clarified that KI was a specific chemical for the extraction of mercury in the sludge and that some oxidation process was essential for the effective extraction of mercury.

Picogram Determination of Methylmercury in Seawater by Gold Amalgamation and Atomic Absorption Spectrophotometry

Abstract An analytical procedure for the determination of total mercury (T-Hg) and methylmercury (CH3Hg) in seawater was described. CH3Hg was extracted with benzene and concentrated by a succession of three partitions between benzene and a cysteine solution. From the last cysteine extract, CH3Hg was transfered into a dithizone chloroform solution. CH3Hg-dithizonate was determined by atomic absorption spectrophotometry (AAS). T-Hg was also determined by AAS after wet combustion of the sample with sulfuric acid and potassium permanganate. The limit of detection was 0.005 ng for CH3Hg, when 3.6 liter of seawater was treated, and the coefficients of variation for CH3Hg 10 ng was about 1%. Seawater from the coast of the Kinki-area, the middle part of the mainland of Japan, seemed to have a T-Hg content of about 10ngl−1 of which nearly 1% was suspected to be the content of CH3Hg.

Fourier transform infrared absorption studies of the sulfhydryl groups in heavy meromyosin

Infrared absorptions of heavy meromyosin solutions were studied in the frequency range of 2600 cm −1 to 1800 cm −1 with a Fourier transform infrared spectrophotometer. An absorption band characteristic of the stretching vibration of sulfhydryl groups was found at about 2565 cm −1. By comparison with the infrared absorption spectrum of a cysteine solution, the absorption band of sulfhydryl groups in heavy meromyosin showed that the absorption intensity is much stronger, the absorption peak shifts to a lower wavenumber and the width of the absorption band is much broadened. These results indicate that the sulfhydryl groups in heavy meromyosin are strongly hydrogen-bonded. The additions of ATP and ADP increased the absorption intensity of the absorption band, suggesting that the hydrogen-bonded structure involving the sulfhydryl groups becomes more strengthened on the binding of ATP and ADP. This indicates that myosin heads change conformation around the sulfhydryl groups during ATP hydrolysis.

Effect of Cysteine on Volatile Development in Dehydrated Onions (Allium cepa L.)

The volatiles developed from dehydrated onion during rehydration with water or 1g/l cysteine solution were trapped on a porous polymer, Tenax-GC, and analyzed by an on-column entrainment gas chromatography procedure. Rehydration temperatures of 22 and 40°C, and four “sampling-trapping” times were investigated. Samples rehydrated with cysteine solution generally gave less total volatiles when the sampling-trapping period was 15, 30 and 60min and more after 2 hours than water rehydrated samples. Propionaldehyde, 1-propanol and 1-propanethiol contents were always higher in samples rehydrated with water. The plausible reactions responsible for the observed effect of cysteine are discussed.

Environmental Hygienic Studies on Mercury Compounds. VI. The Effect of Cysteine on the Transfer of Mercury Compounds to Fetus across the Placenta in Pregnant Rats

Effect of cysteine on the transfer of mercury (methylmercury and inorganic mercury) compounds to fetus across the placenta was examined in pregnant rats. Either of 4 kinds of mercury solutions, methylmercury solution (MM), MM added with cysteine solution (MM-cys), inorganic mercury (HgCl2) solution, and HgCl2 added with cysteine solution (HgCl2-cys), was injected intravenously into pregnant rats at 18 the day of gestation. After 2 hr, they were killed by blood letting from femoral vein and mercury concentration in blood, liver, kidneys, brain, placenta, and fetus was determined. In both placental and fetal mercury, MM-cys group showed significantly higher content that MM group but there was no difference between HgCl2-cys group and HgCl2 group. The ratio of fetal to placental mercury content in MM-cys group was significantly higher than that in MM group. These facts indicate that cysteine was one of the materials which promoted the transfer of methylmercury to fetus across the placenta.

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.

Investigation of the possible use of immobilized urease for decomposing urea in blood plasma

The rate of decomposition of urea in citrated donors' plasma by soluble urease and by urease immobilized by addition to the carboxymethyl ester of cellulose, the 2-(3′-amino-4′-methoxyphenyl) sulfonylethyl ester of cellulose, the diethylaminoethyl ester of cellulose, stained with dichlorotriazine dye, or the grafted copolymer of cellulose and polyglycidylmethacrylate was found to be closely similar. Preparations of immobilized urease can be used repeatedly to decompose urea in citrated donors' plasma. Periodic treatment of these preparations with cysteine solution resulted in a smaller degree of decrease in the enzyme activity of the immobilized urease during repeated use.

Inhibitory Effects of Amine Compounds on the Conversion of Bovine Proparathyroid Hormone to Parathyroid Hormone in vitro

The effects of various amine compounds on the conversion of ProPTH to PTH in bovine parathyroid slices were studied. Bovine parathyroid slices were incubated in Earle's balanced salt solution with or with-out the designated amine compound (pH 7.4) at 37°C for up to 120 minutes. 3H-leucine was added to the incubation medium 30 to 60 minutes before termination of incubation. The incubated tissue slices and medium were homogenized together in 8M urea, 0.2N HCl and 0.01M cysteine solution by Polytron homogenizer. A crude hormonal extract as TCA-powder was prepared through fractionation with organic solvent and salt followed by precipitation of the hormonal peptides with 7.5% TCA. The TCA-powder was further fractionated through CM-cellulose chromatography in order to separate radioactive ProPTH from PTH. Primary, secondary and tertiary amines as well as inorganic ammonium compounds, especially ammonium chloride (pH 7.4), inhibited the conversion of ProPTH to PTH, while quarternary amine, diamine and polyamine did not. Monoamine-induced inhibition of the conversion of ProPTH to PTH was time- and dose-dependent. Such inhibition by amines of the conversion of ProPTH to PTH was reversible in diethylamine and irreversible in tris (OH) methyl-aminomethane.

Studies on the Biosynthesis and Secretion of Parathyroid hormone in Monolayer Cultures of Bovine Parathyroid Cells (I)

We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.

Status of uptake of mercury by the fresh water diatom, Synedra ulna

Synedra, a fresh water diatom, took up mercuric chloride rapidly from the surrounding water into the cells attaining the maximum level during the first 7 h. The comparison of mercury uptake by dividing cells with that of non-dividing, heat killed cells and their silicate shells showed that some factors other than cell-division or photosynthesis were responsible for mercury uptake by Synedra cells. About 20% of the total amount of mercury found in dividing cells was taken up by passive adsorption and could be eliminated by washing with distilled water or medium. About 50% of the mercury could not be eluted even with cysteine solution and was accumulated in the inner part of cells. Synedra took up 0·45 μm filterable or ionic mercury rather than particulate mercury larger than 0·45 μm.

Participation of Cu (II) Ion in the Oxidation of Cysteine with Hydrogen Peroxide

The oxidation of cysteine with hydrogen peroxide was performed in a nitrogen atmosphere and in the presence and absence of Cu (II) ion. The reaction was followed spectrophotometrically by measuring the decreases of cysteine and hydrogen peroxide. One molar hydrogen peroxide could oxidize exactly two molar cysteine and this stoichiometric relation was satisfied under any condition examined. As pH was raised, the oxidation was advanced monotonously. Though addition of Cu (II) to oxygen-free cysteine solution containing hydrogen peroxide enhanced the oxidation significantly, cysteine was oxidized at a fairly rapid speed even in the absence of Cu (II) ion. From the observation on the anaerobic oxidation with hydrogen peroxide, as well as on the autoxidation, a probable mechanism for the copper ion-catalyzed autoxidation of cysteine was deduced.

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl 2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl 2 at a molar ratio of HgCl 2 to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ƒ was similarly sensitive towards HgCl 2, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ƒ and light-induced increase in fluorescence yield were enhanced by the HgCl 2 treatment of chloroplasts. 2. Photooxidation of reduced yeast cytochrome c catalyzed by Photosystem I particles in the presence of plastocyanin was also suppressed by preincubation with HgCl 2. The inhibition was recovered by adding more plastocyanin, but not by addition of the particles, to the reaction mixture. 3. Incubation of reduced plastocyanin with HgCl 2 caused a bleaching of the blue color of the protein, whereas the oxidized protein was resistant to HgCl 2. A similar contrasting difference in sensitivity towards HgCl 2 was found in the Hill reaction in the absence and presence of ferricyanide. 4. Sonic treatment released plastocyanin from the untreated chloroplasts, whereas no plastocyanin was detected in the extract of the HgCl 2-treated chloroplasts. However, plastocyanin could be reconstructed by dialyzing the extract of the HgCl 2-treated chloroplasts against cysteine solution and then CuSO 4 solution. 5. The role of plastocyanin in the photosynthetic electron transport is discussed.

Utilization of the Westöö Procedure for the Determination of Methyl Mercury in Fish by Gas-Liquid Chromatography

Abstract The procedure developed by Westöö for the determination of (mono) methyl mercury in fish is elucidated. The sample is homogenized with water and acidified with HCl and the methyl mercury chloride is partitioned into benzene. The benzene phase is partitioned with a cysteine solution to provide cleanup. The cysteine is then acidified and methyl mercury chloride is again partitioned into benzene and determined by electron capture gas-liquid chromatography. Results are corrected for incomplete partitioning. Recovery of methyl mercury chloride through the partitioning steps is shown to be reproducible. Westoo’s use of mercuric chloride to verify results is substantiated. The normal limit of detection of the method is 0.08 ppm methyl mercury chloride with provisions for detectirg as little as 0.02 ppm.

The mechanism of electroreduction of cystine at the mercury electrode

Potential sweep oscillopolarography of solutions containing cystine and cysteine has been carried out. Mercury cysteinate is formed on the mercury electrode surface in contact with either cystine or cysteine solution at potentials more positive than the reduction potential of cystine. When a cathodic sweep is then applied, the adsorbed mercury cysteinate is reduced, producing a reversible reduction peak. The formation of mercury cysteinate from cysteine is always fast, but its formation from cystine is very slow at low pH values and its rate increases with increasing pH. The mercury in the mercury cysteinate may be in the monovalent or bivalent form but higher surface concentrations of cysteine favor the bivalent form. Heavy metal ions tend to displace mercury from mercury cysteinate shifting the reduction peaks to potentials more negative than that of mercury cysteinate or of the pure heavy metal ion.

The mechanism of electroreduction of cystine at the mercury electrode

Potential sweep oscillopolarography of solutions containing cystine and cysteine has been carried out. Mercury cysteinate is formed on the mercury electrode surface in contact with either cystine or cysteine solution at potentials more positive than the reduction potential of cystine. When a cathodic sweep is then applied, the adsorbed mercury cysteinate is reduced, producing a reversible reduction peak. The formation of mercury cysteinate from cysteine is always fast, but its formation from cystine is very slow at low pH values and its rate increases with increasing pH. The mercury in the mercury cysteinate may be in the monovalent or bivalent form but higher surface concentrations of cysteine favor the bivalent form. Heavy metal ions tend to displace mercury from mercury cysteinate shifting the reduction peaks to potentials more negative than that of mercury cysteinate or of the pure heavy metal ion.

缶詰内面の黒変に関する研究-I

Iron-sulfide blackening of the headspace and liquid parts of tin cans was investi-gated. In the lid and bottom of the C-enameled can, a definite area of iron layer was exposed by scratching the enamel and the tin layers. These cans were filled to 2/3 volume with cysteine solution containing 0 to 4% NaCl at various pH values from 5 to 8. Other cans were 2/3 filled with an extractive of King crab meat containing the cysteine solution. The blackening of the iron surface was observed organoleptically and quantitatively for 6 months at 37°C. In the headspace, the blackening was accelerated by lower pH values and higher concentrations of NaCl. On the contrary, blackening of the liquid parts was remarka-ble at higher pH values and lower concentrations of NaCl. The existence of copper in the crab extractives accelerated the blackening. The acceleration was significant in the liquid part but not in the headspace.

Radiation Chemistry of Foods

When 10‒3m cysteine solution was irradiated in the presence of glucose at the concentration of ten-fold of cysteine, the G-values of products produced from cysteine were similar to those from 10‒3m cysteine solution. On the other hand, the yield of carbonyl compound from glucose was suppressed completely by interaction between cysteine and radicals which are secondarily produced from glucose.Methionine could not suppress the yield of carbonyl compound from glucose, and, G-values of products from methionine varied in comparison with those from solution containing methionine only.From the results using scavenger, it was concluded that oxidation to methionine sulfoxide and cleavage to α-aminobutyric acid was caused by OH and attack, respectively.