Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450 nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.     

Use of pseudosubstrate affinity to measure active protein kinase A

Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.     

Design, synthesis and peroxidase-like activity of 3alpha-helix proteins covalently bound to heme

As a model of artificial peroxidase, de novo designed three-alpha-helix proteins, 3alpha-H9 and 3alpha-H12, covalently bound to Fe(III)-mesoporphyrin IX were synthesized and examined for a peroxidase-like activity. The activity was regulated according to the positions of His residues in the proteins, and the His residues played a role in an acid-base catalytic function.     

Immunoenzyme method for differential assay of free and bound ABA

We performed the immunochemical study of two solid-phase competitive ELISA systems differing in their specificity toward free and bound ABA. A possible application of these systems for the quantification of natural ABA forms without their preliminary separation and purification in a single sample of plant material was demonstrated.     

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.     

Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn²⁺ and readily competes with zinc finger peptides for Zn²⁺ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn₃-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn₃-Sp1 under the conditions of the assay that permit some Zn₃-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn₃-Sp1 by GC1, EDTA readily reacted with Zn₃-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn₃-Sp1 failed to compete for zinc bound in the Zn₃-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn₃-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.     

Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 x 10(3), 44 x 10(3), 38 x 10(3), 11 x 10(3) and 6.5 x 10(3) of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 x 10(3) copies microm(-2). For relative quantitation, we compared wild-type cells to gas1Delta cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.     

Suggested improvements to the standard filter paper assay used to measure cellulase activity

Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized.     

Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estimation of protein in allergen extracts and influence of different parameters on the modified Lowry assay

Protein values of dialysed allergen extracts determined by Lowry, modified Lowry (trichloroacetic acid precipitation of the proteins) and dye-binding assay were compared. The influence of different parameters on the modified Lowry was examined. The reproducibility of the modified Lowry was checked with three independent measurements. For the examination of recovery a constant amount of 6-grass pollen allergen proteins was added to the samples of the standardized human serum albumin prepared for the calibration curve. The samples were measured by modified Lowry. The mean of the ratio between the protein values of the dialysed allergen extracts obtained by modified Lowry and those obtained by classical Lowry was 3.59 (coefficient of variation Cv = 45%). The mean of the ratio between the protein values of the allergen extracts obtained by modified Lowry and dye-binding assay was 1:0.71 (Cv = 31%). Phenol interfered with the modified Lowry. Phenolic allergen extracts showed higher "protein values" than non-phenolic allergen extracts. This influence could be reduced by a second precipitation of the dissolved precipitate. The precipitation of non-phenolic dialysed aqueous allergen extracts was complete after the first trichloroacetic acid precipitation. By incubating samples with the Folin-Ciocalteu's reagent at 55 degrees C in a waterbath, the time necessary for developing the colour could be reduced from 45 min to 5 min. Protein measurements by modified Lowry of a 6-grass pollen allergen extract in three different laboratories showed good reproducibility. For these extract 785 micrograms protein/ml (Cv = 4%) could be measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Employment of a turbidimetric assay system to measure heat-induced protein aggregation

1. 1.|We developed a turbidimetric assay system for quantitation of heat-induced protein aggregation which is presumably caused by protein denaturation.

2. 2.|Rhodanese in 6 M guanidinium chloride was employed in the assay system, because this protein recognizes hydrophobic sites on denatured proteins and aggregates.

3. 3.|Turbidity caused by protein-rhodanase aggregation was recorded at 320 nm by using a u.v./VIS spectrophotometer.

4. 4.|When heated, alcohol dehydrogenase (ADH) aggregates with rhodanese. The increase of ADH-rhodanese aggregation was correlated with the loss of enzymatic activity.

5. 5.|These results indicated that the aggregation was proportional to the extent of ADH denaturation which assumingly caused the loss of ADH activity during heating at 45.5°C.

6. 6.|Similar results were observed when cytosolic proteins from CHO cells were heated at 45.5°C. Heated cytosolic proteins promoted aggregation by complex formation with rhodanese. The aggregation increased with increasing heat dose.

7. 7.|Therefore, the rhodanese assay system can be employed usefully to quantitate the protein aggregation after heat stress.

Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.     

Isolation and identification of phenols in oil of vetiver

Several rare phenols, including 4-vinylphenol, 4-vinyl-2-methoxyphenol and trans-isoeugenol, were identified in oil of vetiver.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum.

Two unusual characteristics of some outer membrane proteins of Rhizobium leguminosarum are described. First, most of the major outer membrane proteins could only be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after lysozyme treatment of the isolated cell envelopes, suggesting a very strong, possibly covalent, interaction of these proteins with the peptidoglycan. These peptidoglycan-associated outer membrane proteins belonged to two distinct groups of immunologically related proteins, groups II and III, as defined by typing with monoclonal antibodies. As members of both groups of proteins could be radioactively labeled by growing cells in the presence of N-[3H]acetylglucosamine, we propose that variation in the apparent molecular weight of the antigens within each group is caused by varying numbers of peptidoglycan subunit residues on only two or three different outer membrane proteins. Second, group III outer membrane proteins, with masses of 35 to 46 kilodaltons, formed oligomers stabilized by divalent cations which resisted complete denaturation in 2% sodium dodecyl sulfate at 100 degrees C. Reconstitution experiments showed that of the divalent cations tested, Ca2+ and, to a lesser extent, Mn2+ and Sr2+ were the best stabilizers.     

New pulsed field gradient NMR experiments for the detection of bound water in proteins

Summary New H₂O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H₂O resonance and pulsed field gradients for coherence selection and efficient H₂O suppression. The method allows for a rapid and sensitive detection of H₂O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly ¹⁵N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.     

The capture proteasome assay: A method to measure proteasome activity in vitro

Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i–β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome–Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO₄ in the proteasome–Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO₄ and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.     

Functional group contributions to the partitioning of phenols between liposomes and water

The temperature dependency of the partitioning of p-alkylphenols and p-halophenols has been determined between dimyristoyl phosphatidylcholine liposomes and 0.15 M NaCl. Partition coefficients increased as a function of temperature below the endothermic phase transition temperature (T_c) of the phospholipid but decreased above this temperature. The transfer process was found to be entropy-dominated below and enthalpy-dominated above the T_c, although large negative entropy changes were observed. Regular changes in the thermodynamic functions, partition coefficients and functional group free energies occurred as a function of the alkyl chain length or size of the halogen substituent below but not above the T_c. This has tentatively been attributed to increased phenol-phospholipid interaction at the higher temperatures. The partitioning of p-fluorophenol behaved in a manner expected of fluorinated compounds, yielding relatively low partition coefficients, but it produced an additional effect of markedly lowering the T_c of dimyristoyl phosphatidylcholine. Good correlations of the partition coefficients in liposomes with those in bulk organic solvents and with molecular size of the solute have been obtained.     

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.     

Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80

Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances.