Isolation and characterization of the 7S protein from glanded cottonseed (Gossypium herbacium)

The major protein from glanded cottonseed has been isolated in a homogeneous form. Its S²⁰ w value at 1 protein concentration is 6S in 1 M NaCl solution. It contains 1 carbohydrate and is free from phosphorus, gossypol (bound or free) and nucleic acid impurities. It consists of atleast seven non-identical subunits. The protein has an ultraviolet absorption maximum at 278 nm and fluorescence excitation and emission maxima at 280 nm and 325 nm respectively. Optical rotatory dispersion and circular dichroism measurements indicate that the protein consists predominantly of Β-structure and random coil. The observed near-ultraviolet circular dichroic bands can be attributed to tyrosine, phenylalanine and tryptophan residues of the protein.     

Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O-methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constantK = 0.4 × 10³ M⁻¹. Data suggests that the lectin could be immobilized without any loss of sugar binding activity     

The chemical modification of cysteine-69 of rat liver fatty acid-binding protein (FABP): a fluorescence approach to FABP structure and function

Summary Hepatic-FABP was labelled at cysteine-69 with the fluorescent environmentally sensitive reporter group AEDANS. The labelled protein had an emission maximum at 465 nm indicating that cysteine-69 was buried in a non-polar environment. The modified protein was still able to bind ligands such as oleic acid, oleoyl CoA and haem. The affinity of AEDANS-FABP for haem was unaltered as compared with the native protein indicating that cysteine-69 must be remote from the ligand binding site. The binding of oleic acid did not significantly perturb the fluorescence emission spectrum of the fluorescent reporter group suggesting that there are not large conformational changes in the region of cysteine-69 on fatty acid binding. The binding of stoichiometric amounts of oleoyl CoA was accompanied by a small fluorescence enhancement which suggests that fatty acyl CoAs may interact with other regions of the FABP molecule not involved in fatty acid binding.Abbreviations FABP Fatty Acid-Binding Protein - AEDANS 5-[2-(Acetyl)aminoEthyl]Aminonaphthalene-1-Sulphonic Acid - AAEDANS 5-[2-(Iodoacetyl-AminolEthyl] aminonaphthalene-1-Sulphonic Acid - DTNB 5,5Dithiobis-(2-Nitrobenzoic acid)     

Effect of chemical modification on struture and activity of glucoamylase fromAspergillus candidus andRhizopus species

The histidine, tyrosine, tryPtoPhan and carboxyl grouPs in the enzyme glucoamylase fromAsPergillus Candidus andRhizoPus sPecies were modified using grouP sPecific reagents. Treatment of the enzyme with diethylPyrocarbonate resulted in the modification of 0.3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase fromAsPergillus candidus andRhizoPus sPecies resPectively. Modification of tyrosine either by N-acetylimidazole or [I¹²⁵]-leads to a Partial loss of activity. Under denaturing conditions, maltose did not helP in Protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the Presence of urea, Photooxidation at PH 9.0, N-bromosuccinamide at PH 4.8 resulted in a comPlete loss of activity. However, the results of exPeriments in the Presence of maltose and at PH 4.8 Photooxidation and N-bromosuccinamide treatment suggested the Presence of two tryPtoPhan residues at the active site. There was a comPlete loss of enzyme activity when 10 and 28 carboxyl grouPs fromAsPergillus candidus andRhizoPus, resPectively were modified. Modification in the Presence of substrate maltose, showed at least two carboxyl grouPs were Present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 tyPes of α-glucosidic linkages namely α-1, 4, α-1, 6 and α-l, 3.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Re‐structuring protein crystals porosity for biotemplating by chemical modification of lysine residues

Protein crystals are routinely prepared for the elucidation of protein structure by X‐ray crystallography. These crystals present an highly accurate periodical array of protein molecules with accompanying highly ordered porosity made of interconnected voids. The permeability of the porous protein crystals to a wide range of solutes has recently triggered attempts to explore their potential application as biotemplates by a controlled “filling” process for the fabrication of novel, nano‐structured composite materials. Gaining control of the porosity of a given protein crystal may lead to the preparation of a series of “biotemplates” enabling different ‘filler’/protein content ratios, resulting in different nanostructured composites. One way to gain such control is to produce a series of polymorphic forms of a given “parent‐protein” crystal. As protein packing throughout crystallization is primarily dominated by the chemical composition of the surface of protein molecules and its impact on protein–protein interactions, modification of residues exposed on the surface will affect protein packing, leading to modified porosity. Here we propose to provide influence on the porosity of protein crystals for biotemplating by pre‐crystallization chemical modification of lysine residues exposed on protein's surface. The feasibility of this approach was demonstrated by the serial application of chemical “modifiers” leading to protein derivatives exhibiting altered porosity by affecting protein “packing” throughout protein crystallization. Screening of a series of modifying agents for lysine modification of hen egg white lysozyme revealed that pre‐crystallization modification preserving their positive charge did not affect crystal porosity, while modification resulting in their conversion to negatively charged groups induced dramatic change in protein crystal's packing and porosity. Furthermore, we demonstrate that chemical modification of lysine residues affecting modified protein packing may be simultaneously performed with the crystallization process: aldehydes generating Schiff base formation with protein's lysine residues readily affected modified protein packing, resulting in altered porosity. Our results demonstrate the feasibility of the use of site directed chemical modifications for the generation of a series of protein crystal exhibiting different porosities for biotemplating, all derived from one “parent” protein. Biotechnol. Bioeng. 2011; 108:1–11. © 2010 Wiley Periodicals, Inc.     

Effect of gossypol on the ultrastructure of rat spermatozoa

Summary Gossypol was found to induce sterility in male rats when administered orally. A reduction in the number of spermatozoa in the epididymis from the gossypol-treated rats was observed when compared to the control animals. An examination of the spermatozoa from the treated rats showed the following ultrastructural modifications: disorganization of the mitochondrial sheath and missing cell membrane from the middle piece, broken cell membrane and missing members of both outer fibers and inner microtubules of the principal piece, and broken cell membrane of the sperm head. Serial mating experiments proved that gossypol-treated males were indeed sterile. The results suggest that gossypol at low concentrations is able to affect the motility of spermatozoa, thus contributing to its contraceptive action.     

Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis

A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.     

Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.     

绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性

气味结合蛋白(odorant binding proteins, OBPs) 在昆虫嗅觉识别中起着重要的作用, 尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors, ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制, 同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum (Meyer-Dür)气味结合蛋白AlucOBP7基因（GenBank登录号: JQ675724）, 并进行了原核表达, 以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及 6种性信息素类似物的结合能力。结果表明： 在10种棉花挥发物中, AlucOBP7能够和2 己酮及水杨酸甲酯有效结合, 结合常数分别为55.13 μmol/L和28.26 μmol/L。在6种盲蝽性信息素类似物中, 4-氧代-反-2-己烯醛和AlucOBP7 具有较强的结合能力, 结合常数为23.14 μmol/L。丁酸乙酯、 丁酸丁酯及己酸己酯也能够和AlucOBP7 有效结合, 但结合能力中等, 结合常数分别为30.58, 39.26和35.81 μmol/L。初步推测, AlucOBP7 可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins, PBPs), 并在感受性信息素和植物挥发物的过程中发挥双重功能。     

Effects of chemical modification of lysine residues on the sweetness of lysozyme

Lysozyme is a sweet-tasting protein with a sweetness threshold value of around 7 microM. To clarify the effect of basicity at the side chain of lysine residues on the threshold values of sweetness, charge-specific chemical modifications such as guanidination, acetylation and phosphopyridoxylation of lysine residues were performed. Sensory analysis showed that the sweetness threshold value of lysozyme was not changed by guanidination, whereas it was increased markedly by acetylation and phosphopyridoxylation. To confirm the importance of the basicity in the lysine residues in detail, purification of acetylated (Ac-) and phosphopyridoxylated (PLP-) lysozymes using SP-ion exchange column chromatography was performed. The threshold values were not changed by modification with fewer than two residues (approximately 7 microM), whereas the threshold values significantly increased to 15 and 34 microM when tetra-Ac and tri-PLP, respectively. Furthermore, sweetness was not detected at 30 microM (hexa-, penta-Ac and tetra-PLP). It should be noted that removal of the negative charges of the phosphate groups in the tri-PLP lysozyme by acid phosphatase resulted in the recovery of sweetness (6.4 microM), indicating that basicity at the position of the lysine residues is responsible for lysozyme sweetness and that strict charge complementarities might be required for interaction to its putative receptor.     

Improving biomaterial properties of collagen films by chemical modification

Films of bovine collagen were chemically modified with the goal of improving their biomaterial properties. The modified films were investigated with respect to their affinity to fibroblast and endothelial cells, as well as their antibacterial properties tested by adhesion of Staphylococcus aureus. Modifications that only change the net charge of collagen, such as acetylation, succinylation, and treatment with glutaraldehyde (all increase the negative charge), and amination with ethylenediamine (EDA), N,N-dimethyl-EDA (DMEDA), or butylamine (all increase the positive charge), did not dramatically alter the mammalian cell attachment to the film. In contrast, derivatization of collagen using methoxypoly(ethylene glycol) (PEG) diminished the attachment of fibroblasts by 98 +/- 1% and of endothelial cells by more than 99% compared to unmodified collagen. Moreover, the rate of growth of fibroblasts dropped by 97 +/- 1% and that of endothelial cells by 88 +/- 3% as a result of PEGylation of collagen. Adhesion of S. aureus cells also plummeted by 93 +/- 2% as a result of this PEGylation. With these antifouling properties, PEG-collagen may be a promising coating material for coronary stents. Subsequent derivatization of PEG-collagen with EDA or DMEDA abolished its mammalian cell-repelling ability, whereas bacterial cell repulsion was partially retained: for example, DMEDA-modified PEG-collagen exhibits up to a 5-fold lower bacterial adhesion than collagen. It is worth noting that a material that allows mammalian cell attachment but reduces bacterial adhesion could be useful as an implant or coating.     

The structure of the TrmE GTP-binding protein and its implications for tRNA modification

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.     

Increased thermal stability of glucose dehydrogenase by cross-linking chemical modification

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65°C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45°C and 75°C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65°C.     

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

岩藻多糖的提取、化学改性、降血糖活性及机理研究进展

岩藻多糖(fucoidans, FU)主要来源于海洋褐藻和海洋无脊椎动物,是一种复杂的硫酸化多糖。FU主要单糖组成为岩藻糖,含有大量硫酸基团,是一种多聚阴离子同型杂多糖。FU具有广泛的潜在健康功效及治疗作用,包括抗肿瘤、调节免疫、抗病毒、降血糖等。FU的化学结构及硫酸基团含量对功能活性具有重要影响,不同提取方法影响FUs的结构组成,而化学改性可以进一步提高其生物活性。因此,本文旨在概述FU的提取、化学改性方法及降血糖活性和机理,展望了FU提取、化学改性、结构及降血糖活性及其他生物活性构效关系方面未来研究方向,为今后的加工和创新利用提供理论参考。