两种萤火虫荧光素酶活性检测方法的一致性比较

目的：比较2种萤火虫荧光素酶活性检测方法的一致性。方法：分别采用化学发光技术（Che）及活体光学成像技术（Bio）,从细胞和动物水平检测在转染以萤火虫荧光素酶为报告基因的载体pCI-AAA-Fluc-neo后不同时间点,萤火虫荧光素酶的表达强度。结果：在细胞和动物水平,萤火虫荧光素酶的表达强度均随时间推移逐步递减。在HepG2细胞,萤火虫荧光素酶表达持续96h,活性从24h的2781±220mV（1．6×10^6±2．3×10^5光子）降至96h的49±3．5mV（6．4×10^4±2．5×10^4光子）。在动物水平得到相似的结果,BALB／c小鼠萤火虫荧光素酶表达持续20d,其活性从1d的16592±409mV（1．9×10^8±3．6×10^6光子）降至20d的798±139mV（3．37×10^5±3．8×10^4光子）。通过一致性检验,2种检测方法在细胞和动物水平的直线回归方程分别为lgChe=1．186·lgBio-3．764（r=-0．937,P〈0．001）和lgChe=0．451·lgBio＋0．64（r=0．915,P〈0．001）;进一步将理论数据与实验数据进行配对t检验,二者无统计学差异（P〉0．05）。结论：2种检测方法是一致的;从整个实验过程来看,活体光学成像技术较化学发光法更为简便、直观,可量化地对同一个体连续检测,减少了个体间差异和实验动物用量。     

Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from <Emphasis Type="Italic">Luciola mingrelica</Emphasis> by the substrates

Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda(em) = 340 nm), but also that of Tyr residues (lambda(em) = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH2) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K(s(ATP)) = 20 and 110 microM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH2. Formation of the complex between the luciferase and LH2 affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.     

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E₂ is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E₂ utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E₂ amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E₂ required to displace 50% of the maximal binding of Cypridina luciferase‐labeled prostaglandin E₂ (B/B₀) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme‐linked immunosorbent assay. Copyright © 2009 John Wiley & Sons, Ltd.     

Stability of immobilized α-chymotrypsin

The thermal of free and immobilized α-chymotrypsin was investigated experimentally and theoretically. The inactivation process of free α-chymotrypsin was analyzed with a kinetic model which included a first- order reaction process and autolysis. The effects of ionic strength, Ca²⁺ concentration, and temperature are discussed here in terms of the estimated kinetic parameters included in this model. The inactivation process of α-chymotrypsin immobilized onto various supports by several methods was investigated. The Contribution of thermal denaturation and autolysis to the inactivation depended upon the method of immobilization. To interpret quantitatively the non-first-order thermal denaturation process of the immobilized enzyme, a model in which the heterogeneity of the immobilized enzyme was taken into account is proposed.     

Development of ultra‐high sensitivity bioluminescent enzyme immunoassay for hepatitis B virus surface antigen using firefly luciferase

Hepatitis B virus (HBV) infection continues to be a global public health concern. Efficient diagnosis of HBV surface antigen (HBsAg) is useful for identification of infection, treatment and prevention of transfusion‐transmitted viral infections. Seronegative window reduction afforded by a highly sensitive measurement methodology is necessary as a small quantity of virus with infection risk exists for the period characterized by undetectable HBsAg following HBV infection. In this study, a bioluminescent enzyme immunoassay (BLEIA) for HBsAg was developed employing firefly luciferase as a labeling enzyme and a two‐step sandwich immunoassay method. The cut‐off value (10 mIU/mL) was 50‐fold more sensitive relative to conventional chemiluminescent enzyme immunoassay based on luminol luminescence involving peroxidase as the labeling enzyme and the identical antibodies. Preliminary clinical data for this BLEIA revealed that the HBV seroconversion panel derived sequentially from HBV‐infected human blood was detected 11 days following window closure from the first bleed, whereas detection occurred 14–25 days following window closure with the three conventional commercial kits. Copyright © 2009 John Wiley & Sons, Ltd.     

Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin–luciferase bioluminescence

Firefly luciferin–luciferase bioluminescence is known for its high quantum yield (41.0 ± 7.4%). Given this high quantum yield, application of this bioluminescence is expected to be useful in the field of clinical diagnostics. The kinetic profile of this bioluminescence exhibits an instant rise (<1 s) and a rapid decay in light emission (decreased to 42% after 5 s). In this study, we applied four enhancers including coenzyme A, inosine5′‐triphosphate sodium salt, sodium tripolyphosphate and potassium pyrophosphate to prolong light emission. When these enhancers were used, luminescence was only decreased to 89, 83, 87 and 82% after 5 s, respectively. These materials modified the kinetic profile of bioluminescence so that the luminescence is more suitable for clinical application. It becomes more suitable because they enable highly sensitive integration and simplification of a device by separating luminescence measurements from dispensing of reagents. Using these enhancers, we then developed a bioluminescent enzyme immunoassay (BLEIA) for hepatitis B virus surface antigen (HBsAg) that employed firefly luciferase as a labeling enzyme. We compared the results obtained from the HBsAg BLEIA method with the conventional chemiluminescent enzyme immunoassay method, and found a satisfactory correlation (r = 0.984, n = 118). Copyright © 2010 John Wiley & Sons, Ltd.     

Conjugation of α-chymotrypsin on a polymeric hydrophilic nanolayer covering magnetic nanoparticles

Magnetic nanoparticles, covered by a polymeric hydrophilic nanolayer containing reactive amino groups, were obtained via Hoffman degradation of the polyacrylamide-coated Fe₃O₄ nanoparticles synthesized by photochemical in situ polymerization, and then conjugated the model enzyme––α-chymotrypsin (CT) by use of EDC· HCl and NHS at room temperatures. The mechanism of photochemical in situ polymerization was briefly proposed in this paper. Superparamagnetic properties were retained for Fe₃O₄ after enzyme immobilization while slightly reducing the value of saturation magnetization. Crystalline structure of Fe₃O₄ after CT immobilization was consistent with that of the freshly prepared Fe₃O₄ by X-ray diffraction (XRD) analysis. The binding capacity was 69 and 61 mg enzyme/g nanogel determined by thermogravimetric (TG) analysis and by standard BCA protein assay, respectively. Specific activity of the immobilized CT was 0.93 U/(mg min), only 59.3% as that of free CT. Thermal stability of CT was improved after being bound to the amine-functionalized magnetic nanogel.     

Covalent binding of α-chymotrypsin on the magnetic nanogels covered by amino groups

A new aminated carrier—magnetic nanogels covered by amino groups, was obtained by Hoffman degradation of polyacrylamide-coated Fe₃O₄ nanoparticles prepared by photochemical polymerization. α-Chymotrypsin (CT) was covalently bound to the magnetic nanogels by use of 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide and N-hydroxysuccinimide at room temperature. Immobilization time, pH value of the reaction mixture and proportion of CT to the magnetic nanogels were investigated to obtain the optimum condition for CT immobilization. The maximal specific activity of the bound CT was determined to be 0.93 U/(mg min), 59.3% of free counterpart. The maximal binding capacity was measured to be 102 mg enzyme/g nanogel. Furthermore, the bound CT exhibited good thermal stability, storage stability and reusability.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

Interleukin-1βconverting enzyme: A novel cysteine protease required for IL-1β production and implicated in programmed cell death

Interleukin-1β converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL-1β, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.     

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

We have developed a bioluminescence‐based non‐destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)‐targeted copepod luciferase (GLuc‐KDEL), respectively, by using multi‐integrase mouse artificial chromosome (MI‐MAC) vector. We have demonstrated that the time‐dependent concentration response curves of ELuc luminescence intensity and WST‐1 assay, and GLuc‐KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant‐treated ELuc‐ and GLuc‐KDEL‐expressing HepG2 stable cell lines. We have clarified that the increase of GLuc‐KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc‐KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage‐associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc‐KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc‐KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.     

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

Single‐molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single‐molecule enzymatic kinetics and provide comparisons to ensemble‐averaged kinetics. To isolate our model enzyme, α‐chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 μm in diameter and 1.35 μm in height. Inside the wells, a protease assay with a profluorescent substrate detects α‐chymotrypsin activity. We hold the concentration of α‐chymotrypsin at 0.39 nM in a given well with an enzyme‐to‐substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge‐coupled device. This method allows for the simultaneous real‐time characterization of hundreds of individual enzymes. We analyze single‐molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Reaction pattern of a novel thermostable α-amylase

A novel thermostable α-amylase, D45 was studied for its reaction pattern on starch hydrolysis. Fine structures of the dextrins and oligosaccharides produced by D45 were determined and compared with those produced by other thermostable α-amylases, Termamyl^®LC (LC) and Termamyl^®SC (SC). Waxy maize starch dispersion was hydrolyzed with LC, SC and D45 at different concentrations to obtain hydrolysates with the same dextrose equivalent value (DE). At DE 13, molecular weight distribution of dextrins produced by D45 displayed a mono-distribution with a peak centered at degree of polymerization (DP) of 7, whereas LC and SC hydrolysates displayed a bimodal-distribution of the molecular weight profiles with one peak centered at DP 5 and the other at DP 34. Thin-layer chromatograms (TLC) showed that DP 2, 3, 5, 6 and 7 were the primary oligosaccharides produced in LC hydrolysate, DP 4–7 in SC hydrolysate, and DP 6–9 in D45 hydrolysate. Comparison of the decrease in the blue color of amylose-iodine complex at 620 nm (blue value) with the increase in reducing value for the hydrolysis of amylose by LC, SC and D45 showed that for an equivalent decrease in blue value, LC and SC produced a higher percentage of reducing sugar than did D45. The results suggest that D45 has a greater degree of random attack (multichain) reaction, whereas LC and SC have more multiple-attack reactions.     

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

α₁‐Antitrypsin (α₁‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α₁‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α₁‐AT, specific mutants of interest, and providing an appended catalog of the >200 α₁‐AT mutants published to date.     

Fluorine nmr studies of poly(N-acryloyl-β-alanine)–α-chymotrypsin conjugates

Fluorine nmr experiments carried out at 51.0 and 94.1 MHz have been used to explore the interaction of the probe molecule p-fluorocinnamate with conjugates formed from α-chymotrypsin and poly(N-acryloyl-β-alanine). The data obtained include enzyme-induced chemical-shift effects, spin-lattice (R₁) and transverse (R₂) relaxation rates, and the rate constant for dissociation of the fluorocinnamate–enzyme complexes. Analysis of the results indicates that while overall molecular tumbling of the enzyme molecule is not greatly changed by attachment of polymers of various sizes, conjugated polymer can appreciably affect the structure of the p-fluorocinnamate binding site. The important variable involved in such structural changes appears to be the amount of polymer present per mole of protein.     

A radioactive assay for the physiological activity of the tryptophan synthetase α subunit in crude extracts of Escherichia coli

A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [³H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.     

A novel colorimetric assay of β‐D‐glucans in basidiomycete strains by alcian blue dye in a 96‐well microtiter plate

Basidiomycete strains synthesize several types of β‐d ‐glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these β‐d ‐glucans in mushroom strains is of great biochemical importance. Because published assay methods for these β‐d ‐glucans present some disadvantages, a novel colorimetric assay method for β‐d ‐glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (～14 nm) in UV‐Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high‐throughput colorimetric assay method on microtiter plates was used for quantification of β‐d ‐glucans in the range of 0–0.8 μg, with a slope of 44.15 × 10^?2 and a limit of detection of 0.017 μg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for β‐1,3‐d ‐glucan. The present assay method exhibited a 10‐fold higher sensitivity and a 59‐fold lower limit of detection compared with the published method with congo red. β‐d ‐glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify β‐d ‐glucans from other biological sources. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1526–1535, 2015     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.