Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo‐Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady‐state and picosecond‐resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N‐dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo‐Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β‐CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature‐dependent circular dichroism studies explore the change in the secondary structure of Apo‐Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Lytic Enzymes toward Aniline-assimilating Rhodococcus erythro-polis AN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN-13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH₄)₂S0₄ fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and 11.0. The molecular weights of C-4 and C-5 were 26,000 and that of C-2 was 36,000, as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-13 than C-4 and C-5.     

Oxysterol Regulation of Estrogen Receptor α-Mediated Gene Expression in a Transcriptional Activation Assay System Using HeLa Cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor α-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17β-estradiol >> genistein >> β-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = β-epoxysitosterol = 7β-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

Polyethylene Glycol–Modified Papain Catalyzed Oligopeptide Synthesis from the Esters of l-Aspartic and l-Glutamic Acids in Benzene

Comparative studies were made of the polymerization of l-aspartic and l-glutamic acid dialkyl esters using polyethylene glycol–modified papain as a catalyst in phosphate buffer (pH 7.5) and in benzene. Changes in the substrate specificity of papain and in the composition of oligomerized products were observed. In the buffer, the diethyl and di-n-propyl esters of l-glutamic acid were sufficiently converted to high molecular weight oligomers with the accumulation of dimer and trimer, but l-aspartic acid esters were very poor substrates. In benzene, l-aspartic acid esters became more reactive than L-glutamic acid esters. In particular, from l-aspartic acid dimethyl ester the product, which was mainly composed of heptamer to decamer, was obtained in a 90% yield. The reaction in benzene required desalted substrates and a small amount of water to proceed extensively.     

A Yeast Bioassay for Androgenic and Anti-Androgenic Compounds Based on the NH2- and COOH-Terminal Interaction of Androgen Receptor

Androgenic compounds induce an interaction between the NH₂- and COOH-terminal regions (N–C interaction) of androgen receptor (AR). We describe a rapid yeast bioassay for androgenic and anti-androgenic compounds based on androgen-dependent β-catenin-enhanced N–C interaction. The bioassay was also effective at detecting compounds that inhibit the N–C interaction in ways that do not involve binding to the ligand-binding domain.     

Structure and Synthesis of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

Dopastin, an inhibitor of dopamine β-hydroxylase of microbial origin, was shown to be N-[2(S)-nitrosohydroxylamino-3-methylbutyl] crotonamide based on chemical, spectroscopic and synthetic studies. The total synthesis of dopastin was completed in 8 steps starting from l-valinol. N-Nitrosohydroxylamino function was introduced through an oxaziran with retention of the absolute configuration in the final product. Thus, the 2S-configuration of dopastin was proved by the total synthesis. Racemic dopastin was also synthesized from isobutyraldehyde in 7 steps.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.