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Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ max of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H 3 PO 4 , indicating negative impact of H 3 PO 4 on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H 3 PO 4 was achieved within 30 and 60 min, respectively. This revealed that H 3 PO 4 has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.

Victor Henri's great contribution to the understanding of enzyme kinetics and mechanism is not always given the credit that it deserves. In addition, his earlier work in experimental psychology is totally unknown to biochemists, and his later work in spectroscopy and photobiology almost equally so. Applying great rigour to his analysis he succeeded in obtaining a model of enzyme action that explained all of the observations available to him, and he showed why the considerable amount of work done in the preceding decade had not led to understanding. His view was that only physical chemistry could explain the behaviour of enzymes, and that models should be judged in accordance with their capacity not only to explain previously known facts but also to predict new observations against which they could be tested. The kinetic equation usually attributed to Michaelis and Menten was in reality due to him. His thesis of 1903 is now available in English., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

The oxidation of cysteine thiol side chains by hydrogen peroxide to afford protein sulfenyl modifications is an important mechanism in signal transduction. In addition, aberrant protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments with molecular specificity. In this review, we trace the history of chemical and biological methods that have been developed to detect protein sulfenylation and illustrate how a recent cell-permeable chemical reporter, DYn-2, has been used to detect and identify intracellular targets of endogenous H2 O2 during growth factor signaling, including the epidermal growth factor receptor. The array of new tools and methods discussed herein enables the discovery of new biological roles for cysteine sulfenylation in human health and disease., (Copyright © 2013 Wiley Periodicals, Inc.)

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595nm) can be identified and corrected by recording absorption spectra in the region of 350-850mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595nm by measuring the sample absorbance at 850nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The growing interest in membrane interactions of amyloidogenic peptides and proteins emanates from the realization that lipids and membranes play important, potentially central, roles in the toxicity and pathological pathways of amyloid diseases. Expanding body of evidence indicates that lipid binding of amyloidogenic peptides and amyloid peptide association with cellular membranes is critical in the onset and progression of amyloid diseases. Advancing the understanding in this field goes hand in hand with application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. This review summarizes experimental approaches and technical aspects employed in recent years for investigating the interaction of amyloid peptides and fibrillar species with lipid bilayers, and the reciprocal contribution of membranes to fibrillation processes and amyloidogenesis.

This unit describes three copper-based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree-Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low-molecular-weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low-molecular-weight contaminants.

Sequencing of the Arabidopsis thaliana genome is complete. The analytical tools for determining gene function by altering and monitoring gene expression are relatively well developed, and are generating large volumes of valuable data. Recent advances in techniques for the analysis of small molecules allow researchers to apply biochemical profiling as another powerful approach to functional genomics and metabolic research.

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

This unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine- and psoralen-conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.

This unit serves as a starting point for exploring the thermodynamic properties of interactions between small molecules and DNA. It covers the determination of simple, apparent association/dissociation constants. The concentration of DNA-bound ligand and free ligand are determined and a binding constant is extracted from these data. Data gathering and curve fitting are discussed.

A design for an improved freeze-quench apparatus is presented. A freeze-quench apparatus has two main parts: the apparatus which mixes the reactants and after a set time sprays the reacting mixture into the quencher, and the quenching apparatus itself. The quenching apparatus is the novel feature of our design and it comprises a gas-flow cryostat mounted directly onto a liquid nitrogen storage Dewar. The gas flow maintains the temperature of a small volume of isopentane, which acts as the freeze-quench agent, at -140 degrees C. (The apparatus can maintain the quenching temperature over the range -190 degrees C to room temperature.) Because of the small size of the cryostat and the much reduced volume of solvent used by this method it is more convenient to use than its predecessors, can be used in the open laboratory, and is safer. Our apparatus is designed for application to electron paramagnetic resonance studies but could be easily modified for use with other spectroscopic techniques.

In the 4 years since Hammes (87) reviewed, in this journal, the application of flow and relaxation techniques to the study of primary processes in chemical reactions these methods have been applied to a wide range of biochemical problems. These applications have been facilitated by advances in stopped flow and temperature jump instrumentation, especially with respect to the range of physical properties that may be observed during a reaction, and the availability of instruments.

Ants of different sexes and castes produce different odorous compounds. In Pheidole fallax Mayr, soldier ants produce an indole base, probably skatole, whereas minor workers produce a trial substance, Males of certain species of Lasius and Acanthomyops produce mixtures of terpenes and an indole base. These mixtures are discharged during mating pheromones. The terpene mixtures are qualitatively similar, but each species produces a blend of distinctive proportions. Citronellol and 2,6-dimethyl-5-hepten-1-ol have been identified in the mixtures by gas chromatography and mass spectrometry.

1. Oxidation of vitamin A acetate with monoperphthalic acid gave 5,6-monoepoxyvitamin A acetate, C(22)H(32)O(3), obtained as pale-yellow crystals, m.p. 65-66 degrees . 2. Saponification of 5,6-monoepoxyvitamin A acetate yielded 5,6-monoepoxyvitamin A alcohol, which was readily oxidized with manganese dioxide to 5,6-monoepoxyvitamin A aldehyde, obtained as yellow crystals, m.p. 101-102 degrees . It was the most stable of all the epoxy compounds studied. 3. Treatment of the 5,6-epoxy compounds with ethanolic hydrochloric acid gave the corresponding 5,8-epoxy (furanoid) compounds. 5,8-Monoepoxyvitamin A aldehyde was obtained as crystals, m.p. 104-105 degrees , but was very unstable. 4. Crystalline semicarbazones and phenylhydrazones with constant melting points and characteristic spectra were prepared from 5,6- and 5,8-monoepoxyvitamin A aldehyde. 5. Reduction of 5,6- and 5,8-monoepoxyvitamin A aldehyde with lithium aluminium hydride gave the corresponding 5,6- and 5,8-monoepoxyvitamin A alcohol. 6. 5,6- and 5,8-Monoepoxyvitamin A aldehyde were fed to vitamin A-deficient rats, and the compounds obtained from the livers of rats were indistinguishable from the reduction products obtained with lithium aluminium hydride. 7. The structures of the epoxy compounds were confirmed by their chromatographic behaviour, elemental analyses, ultraviolet-, visible- and infrared-absorption spectra and nuclear-magnetic-resonance spectra.

1. (+)-Mollisacacidin [(+)-3',4',7-trihydroxy-2,3-trans-flavan-3,4-trans- diol] is converted by autoclaving into the optically active free phenolic 2,3-trans-3-4-cis (12% yield), 2,3-cis-3,4-trans (11%) and 2,3-cis-3,4-cis (2.8%) diastereoisomers through epimerization at C-2 and C-4. 2. The relative configurations of the epimeric forms were determined by nuclear-magnetic-resonance spectrometry and paper ionophoresis in comparison with synthetic reference compounds, and was confirmed by chemical interconversions. 3. From this a scheme of epimerization is inferred and their absolute configurations are assigned as (2R:3S:4S), (2S:3S:4R) and (2S:3S:4S) respectively from the known absolute configuration (2R:3S:4R) of (+)-mollisacacidin.

1. The growth factor from Mycobacterium phlei is now named mycobactin P to distinguish it from related but chemically distinct growth factors from other species of mycobacteria. It is shown to comprise four closely similar chemical entities differing only in the fatty side chain. 2. The structure of the main component (85%) of mycobactin P has been established and the absolute configuration of all asymmetric centres determined. 3. Ferric mycobactin P has been crystallized; its structure is discussed. 4. Evidence is given for regarding ferric mycobactin P as a member of the class of compounds known as sideramines, though the mycobactins are more specialized than other sideramines.