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1.
Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.  相似文献   
2.
郑蒙蒙  韩颖  康经武 《色谱》2019,37(12):1282-1290
发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20 μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×105个人急性早幼粒白血病(NB4)细胞与人急性T细胞白血病(Jurkat T)细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2489个和2572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。  相似文献   
3.
The extent to which drugs combine with trypsin is influenced by the interaction of tartrazine and trypsin, which may cause overdose or underdose of drugs. Therefore, the interaction of tartrazine and trypsin is investigated by methods of spectrometry in this paper. The binding rate of tartrazine to trypsin is 80.95–95.71% at 310?K, and Hill’s coefficients are almost 1. The effect of tartrazine on trypsin structure was studied by synchronous and circular dichroism. The results showed that the binding of tartrazine and trypsin induced the conformational change of trypsin, and quenched the endogenous fluorescence in trypsin. The results of molecular docking revealed that tartrazine is located in the catalytic active site of trypsin, and is consistent with that of experimental calculation.  相似文献   
4.
ABSTRACT

The interactions between rutin and trypsin were investigated by UV-Vis absorption, CD, fluorescence, resonance light-scattering spectra, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. Rutin effectively quenched the intrinsic fluorescence of trypsin via static quenching. The enthalpy change and entropy change were estimated to be ?8.23 kJ·mol?1 and 53.66 J·mol?1·K?1 according to the van't Hoff equation. The process of binding rutin to trypsin was a spontaneous molecular interaction procedure. This result indicates that hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The conformation of trypsin was discussed by CD, synchronous, and three-dimensional fluorescence techniques.  相似文献   
5.
In this investigation, methods based on on-probe enzymatic cleavage matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analyses have been developed, allowing the rapid assignment of phosphorylation sites within phosphoproteins. The procedures involved robotic sample deposition of a phosphoprotein, such as intact bovine β-casein, on stainless steel or gold MALDI plates, on-probe proteolysis with trypsin for 10–180?s at 37°C, on-probe dephosphorylation for 1–10?min at 37°C with alkaline phosphatase, followed by differential mass spectrometry with peptide mass mapping. The dephosphorylation conditions were initially optimized using in-solution tryptic digestion of the phosphoprotein performed in the presence of MS-compatible anionic surfactant sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate. Two methods of trypsin deactivation were investigated, cooling and quenching by acidification, which resulted in the surfactant either staying intact or becoming cleaved, respectively. Since the surfactant had no detrimental effects on dephosphorylation of phosphopeptides, the acidification and neutralization steps were not included in the final analytical method. A protocol, comprising on-probe tandem, surfactant-aided proteolysis for 3?min followed by on-probe dephosphorylation for 10?min was thus established, allowing the rapid identification of location and sequence of phosphopeptides within a phosphoprotein by these procedures.  相似文献   
6.
为检验人尿胰蛋白酶抑制剂(UTI)两个结构域中的盐键的稳定性对UTI热变性可逆性的影响,将UTI中的Lys121突变成Ile以中断Glu69与Lys121之间形成的盐键,并命名为?UTI.然后对?UTI进行表达纯化和热解折叠实验.结果显示,?UTI在大肠杆菌中成功表达,纯化复性后经园二色谱测定三级结构与UTI基本相似,酶学测定表明恢复胰蛋白酶抑制剂活性.热变性实验表明:在pH低于7.4的缓冲溶液中?UTI的热解折叠是可逆的,与UTI的结果(pH<4.2)相比,pH值提高了3.结合以前对UTI在酸性条件下可逆热解折叠而在中性与碱性条件下不可逆热解折叠的机制的研究结果,认为多结构域蛋白解折叠的曲线除考虑各个结构域单独形成的多个S曲线的叠加外,还要考虑到结构域之间相互作用强度的影响.  相似文献   
7.
8.
The objective of this study was to characterize the fractionation profile of casein hydrolysates obtained with polysulfone hollow fiber ultrafiltration membranes. The two-step ultrafiltration process developed by Turgeon and Gauthier [J. Food Sci., 55 (1990) 106] was used: a caseinate solution was submitted to proteolysis with chymotrypsin or trypsin, and the reaction mixture (RM) was subsequently ultrafiltered using a 30 kDa (MWCO) hollow-fiber polysulfone membrane. The total hydrolysate permeating from this first step was further fractionated using a 1 kDa (MWCO) membrane, producing the mixture of polypeptides (retentate) and the amino acid fraction (permeate). The effect of enzyme specificity and of membrane retentivitiy on the total composition (total nitrogen, fat, lactose, minerals) and amino acid profile of the fractions was studied. The overall composition of the fractions was not significantly affected by the nature of the enzyme but the degree of hydrolysis and the molecular weight distribution profile analyses showed a marked effect of the enzyme specificity, with trypsin giving a larger proportion of small peptides (< 200 Da) in the mixture of polypeptides. Amino acid profile analyses provided useful information on the phenomena governing the fractionation of amino acids with a polysulfone membrane: (1) the target amino acids of the enzyme are concentrated in the permeate as a result of their presence in all peptides produced by hydrolysis, (2) polar amino acids are retained by the membrane, (3) non-polar amino acids are not selectively rejected by the membrane. Our results suggest that the charge/hydrophobicity balance of the peptides produced is the predominant factor determining the fractionation of casein hydrolysates.  相似文献   
9.
田嘉嘉  陈爽  李东辉 《应用化学》2006,23(12):1313-0
荧光各向异性;罗丹明;蛋白酶;胰蛋白酶;胃蛋白酶;菠萝蛋白酶;蛋白酶抑制剂  相似文献   
10.
When trypsin reacts with Herring sperm DNA (hsDNA), Salmon sperm DNA (sDNA), and Calf thymus DNA (ctDNA) to form a complex, the resonance Rayleigh scattering (RRS) was remarkably enhanced and new RRS spectra appear. These new spectra have similar characteristics of RRS spectra. The maximum RRS peaks are at 307 nm (hsDNA, sDNA) and 290 nm (ctDNA), and other peaks are at 350 nm. The scattering intensity is proportional to the concentration of DNA or trypsin; so this intereaction can be used to determine trypsin using DNA or DNA using trypsin. In the determination of DNA using trypsin, the linear ranges for hsDNA, sDNA, and ctDNA are 0–2.3, 0–2.5, and 0–1.9 μg·mL−1, and the detection limits are 0.4, 0.7, and 1.1 ng·mL−1, respectively. In the determination of trypsin using hsDNA, the linear range is 0–30.0 μg·mL−1, and the detection limit is 39.0 ng·mL−1. In this paper, the intereaction conditions were optimized. The affecting factors, chemical properties of the complex, and the composition ratio of trypsin with DNA were investigated. Using trypsin as RRS probe, a sensitive method for the determination of trace amounts of DNA was developed. Translated from Chemical Journal of Chinese Universities, 2006, 27(3) (in Chinese)  相似文献   
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