用碱水解的方法测定蛋白质的消光系数

在蛋白质结构与功能的研究中,有时蛋白质溶液的浓度是一个重要的参数.紫外吸收法是测定蛋白质溶液浓度最为常用的方法,而已知蛋白质的消光系数是用紫外吸收法准确测定蛋白质溶液浓度的前提条件.在0.1 mol/L NaOH溶液中,蛋白质发生碱性水解,因而蛋白质溶液可以看作是色氨酸和酪氨酸的二元体系.以此为依据,给出了用碱水解的方法测定蛋白质消光系数的方法.这一方法操作步骤简便易行,蛋白质消光系数的计算公式简单明了.用这一碱水解的方法分别测定了几种氨基酸组成不同的蛋白质的消光系数,与文献数据对照,得到了令人满意的结果,测定误差均小于±5％.     

A procedure for total protein determination with special application to allergenic extract standardization

A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.     

Protein determination in the presence of glycerol by the Lowry procedure. I. Influence of reaction temperature and alkali.

The influence of glycerol on the Lowry determination of bovine serum albumin (maximum, 20 μg of BSA/sample) was studied. A modified procedure was introduced by applying low temperature during copper complex formation, followed by 37°C after the addition of the Folin reagent. This ensured good accuracy in absorbance readings at the highest glycerol concentration of 22%. However, a strong interference resulting from storing the samples in 0.2 n NaOH could not be overcome.     

Protein precipitation assay for quantitation of tannins: determination of protein in tannin-protein complex

A protein precipitation method for the determination of tannins has been developed. The protein in the tannin-protein complexes was measured using the ninhydrin assay of amino acids released by alkaline hydrolysis of the complex. Standard protein and the complex were hydrolyzed with 13.5 N NaOH at 120 degrees C for 20 min and the amino acids released were measured with ninhydrin. Tannins did not interfere in the determination of protein by ninhydrin assay. The bovine serum albumin (BSA) precipitated (y; mg) increased linearly with increase in tannic acid (x) from 0.2 to 0.9 mg (y = 2.598x - 0.258). The protein precipitation capacities (mg BSA precipitated/g dry wt) measured by the method for young and mature leaves of oaks were Quercus incana (young, 42.21; mature, 79.51), Q. ilex (young, 1.86; mature, 1.86), and Q. semecarpifolia (young, 733.54; mature, 304.32). The method can provide valuable information on the mechanisms of protein-tannin interactions and nutritional and physiological significances of tannins.     

Elimination of insulin-like activity present in certain batches of crude bovine serum albumin by trypsin treatment

A comparative study was carried out in order to determine which of the most commonly used alkalies for protein hydrolysis in tryptophan determination gave the best results. Hydrolyses were performed with 2.5 and 4 n Ba (OH)₂, 4 and 10 n NaOH, 5 n NaOH containing 5% SnCl₂, and with 4 n LiOH, not previously reported for use. The effect of temperature and hydrolysis time on the measured tryptophan content was also determined. Based on results obtained with lysozyme and with seven high protein preparations 4 n LiOH gave the best results. A temperature of 145°C was selected as the most convenient temperature since maximum tryptophan values were obtained with 4–8 h. The hydrolysis time required was inversely related to the protein content of the preparation. Lysozyme, casein, bovine plasma protein, and dehydrated whole egg gave maximum tryptophan content after 4 h hydrolysis while skimmed milk powder, rice flour, wheat flour, and wild legume flour required 8 h hydrolysis.     

Evaluation of alkali treatment for biodegradation of corn cobs byAspergillus niger

Anstract Effect of NaOH pretreatment on the biodegradation of corn cobs for the production of cellulase and protein was studied usingAspergillus niger. Delignification of cobs with NaOH remarkaby increased the production of cellulase and protein. Treatment of cobs with 2% NaOH was found to be the best with respect to their susceptibility to biodegradation for maximum production of cellulose 1,4-β-cellobiosidase, cellulase, β-glucosidase soluble protein and crude protein; this also led to the highest protein recovery, maximum cellulose utilization and also for the maximum degradation of substrate.     

Electrochemiluminescence of palmatine being oxidized by electrogenerated hydroxyl radical and its analytical application

A strong electrochemiluminescence (ECL) of palmatine in NaOH medium was observed at a vaseline‐impregnated graphite anode. The ECL production could be described as follows: hydroxyl radical (OH^?) was generated via the oxidation of hydroxyl group (OH^‐) in NaOH medium, and the formed OH^? subsequently oxidized palmatine base converted from palmatine in NaOH medium to the excited state oxypalmatine (oxypalmatine*). As the oxypalmatine* went back to its ground state, a stronger chemiluminescence was produced. Based on the ECL of palmatine, an ECL method for the determination of palmatine was proposed. An ECL signal of palmatine in NaOH solution was obtained by applying direct current of 15 mA to the vaseline‐impregnated graphite anode. The ECL intensity was rectilinear with palmatine concentration in the range of 8.0 × 10^?7 to 2.0 × 10^?5 mol l^?1 and the limit of detection (signal‐to‐noise = 3) was 3 × 10^?7 mol l^?1. The proposed method was applied to the determination of palmatine in pharmaceutical preparations. Copyright © 2010 John Wiley & Sons, Ltd.     

The applicable condition of Magos' method for mercury measurement under coexistence of selenium

Delayed and incomplete release of Hg⁰ was reported in liver, blood, and spleen, but not in kidneys and brain, of mice simultaneously administered HgCl₂ and Na₂SeO₃ for both inorganic and total mercury determination by Magos' method. This problem was overcome by the treatment of the tissue homogenate with an equal volume of 45% NaOH containing 1% cysteine·HCl at 40°C for more than 30 min and the use of the area under the peak of the mercury release curve on calculaton. In the case of administration of methylmercuric chloride instead of mercuric chloride, the influence of coexistent selenium was not observed for mercury determination by Magos' method in mice within 24 h after dosing.     

Practice of solid-phase extraction and protein precipitation in the 96-well format combined with high-performance liquid chromatography–ultraviolet detection for the analysis of drugs in plasma and brain

C₁₈ Empore 96-well extraction disc plates have been employed for the analysis of three drugs with different polarities in plasma in conjunction with HPLC–UV, rufinamide, ICL670 and an anticonvulsant agent (AA1) in an early stage of development. With the most polar compound (AA1), ion-pair extraction at pH 12 was applied. The method developed for the assay of AA1 in plasma was applied to its determination in brain using an Oasis HLB plate following homogenisation in a pH 7.4 buffer and protein precipitation with NaOH–ZnSO₄, thereby saving time for method development. Protein precipitation in the 96-well format with filtration of the precipitate was applied to the determination of ICL670, a highly protein-bound compound (>99.5%), with a good recovery (78%). Reversed-phase chromatography was applied using a short 5 cm column packed with 3 μm particles for the determination of ICL670 and AA1 and two parallel columns (15 cm long) for the determination of rufinamide. The methods were used routinely, one plate per analysis day being processed, resulting in increase in sample throughput and saving in solvents.     

Effects of urea and sodium hydroxide on the molecular weight and conformation of alpha-(1-->3)-D-glucan from Lentinus edodes in aqueous solution

The weight-average molecular weight (Mw) and intrinsic viscosity ([eta]) of the alpha-(1-->3)-D-glucan (L-FV-II) from Lentinus edodes in 0.5 and 1.0 M NaOH aqueous solution containing urea, were studied by light scattering and viscometry. The Mw value of the glucan decreased with increase of the urea and NaOH concentration. A strong intermolecular hydrogen bonding confers water-insolubility on the glucan, but NaOH and especially urea, broke this hydrogen bonding leading to enhanced water-solubility. Use of 1.0 M urea-1.0 M NaOH as solvent broke not only intermolecular hydrogen bonds but also partial covalent bonds of the alpha-glucan in aqueous solution, resulting in a decrease of Mw and [eta]. The urea and NaOH concentrations, storage time with stirring, and mode of preparation of the polysaccharide in aqueous solution significantly affected the determination of Mw and [eta]. The dependences of specific rotation and fluorescence emission ratio of a probe on urea concentration showed that a change in the molecular conformation of the alpha-glucan in 0.5 M NaOH aqueous solution containing urea occurred in the range 0.4-0.6 M urea. The 0.5 M urea-0.5 M NaOH aqueous solution is a suitable solvent for the glucan, and the Mw and [eta] values obtained were 5.21 x 10(5) and 148 cm3 g(-1), respectively. Degradation of the glucan was obvious after storage for 15 months.     

New aspects of the extraction of chitin from squid pens

In this work, we studied the extraction of beta chitin from squid pens. The characterization by ICP of the raw material confirmed the significant absence of calcium carbonate and then the possibility to definitely avoid a step of demineralization responsible for chain degradation. The kinetics studies associated with the extraction of lipid parts showed the important role played by the latter structures on the limitation of the protein extraction by a simple treatment in an aqueous solution of 1 M NaOH. The role of various parameters such as the size of the particles, time and number of steps, and the concentration in sodium hydroxide was studied. The determination of the molecular characteristics (molecular weight and degree of acetylation) allowed us to define the optimal conditions of deproteinization in relation with the best preservation of these characteristics.     

Structure of aqueous solutions of microcrystalline cellulose/sodium hydroxide below 0 degrees C and the limit of cellulose dissolution

The aim of the paper is to investigate the structure of solutions of microcrystalline cellulose in NaOH/water mixtures and to determine the limit of cellulose solubility. The binary NaOH/water and the ternary cellulose/NaOH/water phase diagrams in the area of cellulose dissolution (7-10% NaOH below 0 degrees C) are studied by DSC. The NaOH/water binary phase diagram has a simple eutectic behavior. Because of the existence of this eutectic structure, it is possible to measure the influence of the addition of pure low molar mass microcrystalline cellulose. This shows that a minimum of four NaOH molecules should be linked to one anhydroglucose unit to allow for the dissolution of microcrystalline cellulose. The proportions between bound Avicel, NaOH, and water molecules as a function of cellulose concentrations are calculated. A tentative explanation about the origin of the dissolving power of NaOH/water is given.     

An enzyme-immobilized microplate for determination of linamarin for large number of samples

Summary An enzyme-immobilized microplate for determination of linamarin was prepared by covalently linking cassava leaf linamarase to the microplate. For linamarin determination, cassava roots were homogenised in 0.1 Mo-phosphoric acid and the filtrate adjusted to pH 6 with NaOH prior to adding into the wells. The cyanide released was then determined spectrophotometrically. One nmol linamarin can be detected. The microplate method is suitable for analysis of large number of samples and is useful for screening purposes.     

A Coomassie blue-binding assay for the microquantitation of immobilized proteins

A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures.     

Mutagenic reactivities of 3,4-dinitrobiphenyl derivatives

3,4-Dinitrobiphenyl derivatives were mutagenic in Salmonella typhimurium TA98, TA98/1,8-DNP6 and in TA98NR. We describe here the specific reactivity of 3,4-dinitrobiphenyl derivatives with diluted sodium hydroxide solution and the determination of the amounts of released nitrous ion. 3,4-Dinitrobiphenyl derivatives begin to release nitrous ions when treated with NaOH solution at a concentration of 10(-3) N. The behavior of 4NQO and o-dinitrobenzene was the same as that of 3,4-dinitrobiphenyl derivatives. The residues of 3,4-dinitrobiphenyl derivatives, after releasing nitrous ions, were estimated to be hydroxy-nitrobiphenyls, as by GC/MS, we found the formation of o-nitrophenol in the reaction mixture of o-dinitrobenzene with aqueous NaOH solution. 3,4,4'-Trinitrobiphenyl, 3,4,3',4'-tetranitrobiphenyl and 4NQO had reduced mutagenic potency in Salmonella typhimurium TA98 following treatment with diluted NaOH. In order to elucidate the ultimate forms of 3,4-dinitrobiphenyl derivatives, we investigated the reaction of o-dinitrobenzene as a basic model substance of 3,4-dinitrobiphenyl, with nucleic bases in the presence of NaOH in nonaqueous solvent. o-Nitrophenyl guanine and adenine adducts were obtained.     

Alkali treatment and fermentation of straw for animal feed

The feed value of annual ryegrass straw was improved by treatment with various concentrations of NaOH or NH₃ followed by fermentation of the treated straw with a mixed culture of Cellulomonas sp. and Alcaligenes faecalis. Laboratory feeding trials with voles showed that NaOH or NH₃ treatment considerably increased the feed efficiency of straw, but apparently gave a poorly palatable product. Fermentation tended to decrease the in vitro rumen digestibility (IVRD) of alkali-treated straw. The fermentations were carried out aerobically on a semisolid straw matrix having 11–86% moisture. Treatment by both NaOH and NH₃ increased the IVRD of straw. NH₃ also increased the nitrogen content in straw. The optimum condition for alkaline treatment of the straw was 4–6% NaOH for 1 hr or with 3% NH₃ for four weeks at room temperature. A minimum of 63% moisture was needed for significant fermentation of the straw. The combined effects of NaOH treatment and fermentation more than doubled crude protein, doubled crude fat, and increased IVRD by 75%. The NH₃ plus fermentation treatment tripled crude protein, doubled crude fat, and increased IVRD by 60%. Acetic acid was the main volatile fatty acid in the fermented straw.     

Single cell protein production from bagasse pith pretreated with sodium hydroxide at room temperature

Summary Previous publications have revealed that a pretreatment of lignocellulosic wastes is necessary if they are to be employed as the hydrocarbon source of single cell protein production. A hot alkaline treatment is the most common.We have treated sugar cane bagasse pith with 1% NaOH solution at room temperature, at a NaOH/pith ratio of 10%. Different contact times were used in the experiments. The shortest contact period required for maximum protein production was 24 h at 25° C. A mixed culture of Cellulomonas sp. and Bacillus subtilis was used in the experiments. The values obtained for hemicellulose and cellulose in the treated pith did not differ greatly from those of untreated pith, in contrast the amount of lignin was 33% lower in the treated pith. The effect of reutilization of the alkaline liquor used for the pretreatment of pith upon protein production was also investigated. With four recyclings, there was a NaOH saving of 34.4 kg per 100 kg produced protein as compared to when the liquor was only used once.The quality of the resulting effluents, as measured by the chemical oxygen demand (COD), proved to be very similar for both types of treatment.     

分泌性的糖蛋白Wnt5a对永生化小鼠黑素细胞系melan-a细胞产黑素的影响

目的：探讨分泌性的糖蛋白Wnt5a对永生化小鼠黑素细胞系melan-a细胞产黑素的影响。方法：用带有Wnt5a基因的腺病毒及对照组腺病毒作为载体感染体外培养的melan．a黑素细胞;MTT法测定细胞增殖率;体外氧化DOPA反应法测定酪氨酸酶活性;NaOH法测定黑素含量;RT-PCR方法检测melan-a细胞中MITF的表达。结果：与AdGFP对照组相比,AdWnt5a处理组melan-a细胞的增殖率明显降低（P〈0．01）;DOPA反应法和NaOH法检测结果发现,Wnt5a能显著降低melan-a细胞内酪氨酸酶活性（P〈0．01）以及黑素含量（P〈0．05）;RT—PCR结果表明,wnt5a显著下调melan—a细胞内MITF的表达（P〈0．01）。结论：以上结果显示,AdWnt5a处理组melan-a细胞的增殖率、酪氨酸酶活性、产黑素的量及MITF的表达均有所下降。实验结果提示,Wnt5a能有效抑制melan-a细胞产黑素的能力,并且其作用机制可能与下调MITF的表达有关。     

THE EFFECT OF RENNIN UPON CASEIN : II. FURTHER CONSIDERATION OF THE PROPERTIES OF PARACASEIN.

The properties of the paracasein and casein preparations studied are compared in Table VI. See PDF for Structure I. Casein retains its characteristic solubility in NaOH: (1) after being exposed to a high degree of alkalinity during its preparation, (2) when recovered from partially hydrolyzed solutions in NaOH, and (3) after being kept for a prolonged time at the isoelectric point at 5°C. II. It follows from I, that: (1) paracasein is not identical to casein modified by an excess of alkali, and that (2) this protein was not produced from casein by a partial hydrolysis of the latter in presence of NaOH.     

Electroimmunoassay of rat apolipoproteins A-I, A-IV, and E. A procedure for sample treatment to increase the sensitivity in diluted fractions

Methods for the quantitative determination of rat apolipoproteins A-I, A-IV, and E by electroimmunoassay are described. Apolipoproteins present in diluted samples of biological fluids (approx. 2 ml) were concentrated by precipitation with deoxycholate and trichloroacetic acid. The protein pellets were solubilized in 0.1 ml of 0.5 M NaOH and these samples were delipidated with tetramethylurea and assayed. This protocol enables the measurement of apolipoprotein concentrations that are at least 10 times lower than normally detectable; 0.2 micrograms of apolipoprotein A-IV, 0.2 micrograms of apolipoprotein A-I, and 0.8 micrograms of apolipoprotein E can be easily detected in samples of 2 ml.