Thermodynamics for the interaction of epsilon-dinitrophenyl-L-lysine and bovine colostral anti-dinitrophenyl immunoglobulin G2.
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ABSTRACT:
SUBMITTER: Mukkur TK
PROVIDER: S-EPMC1185746 | biostudies-other | 1978 Jul
REPOSITORIES: biostudies-other
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Project description:Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.
| S-EPMC1186360 | biostudies-other
Project description:ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.
| S-EPMC4135771 | biostudies-literature
Project description:PurposeTo determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii.MethodsA. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration.ResultsThe toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL.ConclusionspɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.
| S-EPMC8742527 | biostudies-literature
Project description:ε-Poly-lysine (ε-PL) is an uncommon cationic, naturally-occurring homopolymer produced by the fermentation process. Due to its significant antimicrobial activity and nontoxicity to humans, ε-PL is now industrially produced as an additive, e.g. for food and cosmetics. However, the biosynthetic route can only make polymers with a molecular weight of about 3 kDa. Here, we report a new chemical strategy based on ring-opening polymerization (ROP) to obtain ε-PL from lysine. The 2,5-dimethylpyrrole protected α-amino-ε-caprolactam monomer was prepared through cyclization of lysine followed by protection. ROP of this monomer, followed by the removal of the protecting group, 2,5-dimethylpyrrole, ultimately yielded ε-PL with varying molecular weights. The structure of this chemosynthetic ε-PL has been fully characterized by 1H NMR, 13C NMR, and MALDI-TOF MS analyses. This chemosynthetic ε-PL exhibited a similar pKa value and low cytotoxicity as the biosynthetic analogue. Using this new chemical strategy involving ROP without the need for phosgene may enable a more cost effective production of ε-PL on a larger-scale, facilitating the design of more advanced biomaterials.
| S-EPMC6054053 | biostudies-literature
Project description:ε-Poly-L-lysine (ε-PL) is a natural amino acid polymer produced by microbial fermentation. It has been mainly used as a preservative in the food and cosmetics industries, as a drug carrier in medicines, and as a gene carrier in gene therapy. ε-PL synthase is the key enzyme responsible for the polymerization of L-lysine to form ε-PL. In this study, the ε-PL synthase gene was overexpressed in Streptomyces albulus CICC 11022 by using the kasOp∗ promoter and the ribosome binding site from the capsid protein of phage ϕC31, which resulted in a genetically engineered strain Q-PL2. The titers of ε-PL produced by Q-PL2 were 88.2% ± 8.3% higher than that produced by the wild strain in shake flask fermentation. With the synergistic effect of 2 g/L sodium citrate, the titers of ε-PL produced by Q-PL2 were 211.2% ± 17.4% higher than that produced by the wild strain. In fed-batch fermentations, 20.1 ± 1.3 g/L of ε-PL was produced by S. albulus Q-PL2 in 72 h with a productivity of 6.7 ± 0.4 g/L/day, which was 3.2 ± 0.3-fold of that produced by the wild strain. These results indicate that ε-PL synthase is one of the rate-limiting enzymes in ε-PL synthesis pathway and lays a foundation for further improving the ε-PL production ability of S. albulus by metabolic engineering.
| S-EPMC7188835 | biostudies-literature
Project description:Epsilon-poly-L-lysine (epsilon-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces epsilon-PL-degrading enzymes, the original chain length of the epsilon-PL polymer product synthesized by epsilon-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the epsilon-PL-degrading enzyme (PldII), which plays a central role in epsilon-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an epsilon-PL of unchanged polymer chain length, demonstrating that the length is not determined by epsilon-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of epsilon-PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during epsilon-PL fermentation, rather than the inhibition of the epsilon-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.
| S-EPMC2935060 | biostudies-literature
Project description:Microbial secondary metabolites produced by actinomycetes are important natural products widely applied to control plant diseases. A variety of actinomycetes were isolated from soil samples collected from Tianzhu Mountain in Shenyang, China. A Streptomyces strain Shenyang Tianzhu (STZ) exhibits effective antiviral activity against Tobacco mosaic virus (TMV). The isolate was identified as Streptomyces ahygroscopicus based on its cultural, morphological, physiological, biochemical characteristics as well as the phylogenetic analysis using 16S rRNA sequences. To obtain the pure anti-TMV compound from Streptomyces STZ, the culture broth was subjected to Amberlite IRC-50 ion-exchange resin, SX-8 macroporous adsorption resin and Sephadex G-25 gel column chromatography. The purified active compound was confirmed to be ε-poly-l-lysine (ε-PL), with molecular mass in the range of 3454⁻4352 Da by structural analysis with infrared (IR), matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF), thin-layer chromatography (TLC) and high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR). The protective and curative effects of the purified compound ε-PL were tested and the results showed that the compound exhibited significant protective and curative activity against TMV. The potential application of ε-PL as an efficient anti-plant virus agent was expected.
| S-EPMC6471872 | biostudies-literature
Project description:A series of N-(N-dinitrophenylaminoalkyl)maleimides were sythesized with alkyl-chain lengths of two, four and six carbon atoms. When these compounds reacted with the thiol group of mercaptalbumin, the tryptophan fluorescence of the protein was quenched. This change in fluorescence was used to determine the rate of reaction of the Dnp (dinitrophenyl)-maleimides with mercaptalbumin. The second-order rate constants were similar to those observed in reactions between low-molecular-weight thiol compounds and maleimides. When N-(N-Dnp-aminoalkyl)succinimidomercaptalbumins were added to univalent fragments of anti-Dnp antibody the antibody fluorescence was quenched. Florescence-quenching titrations showed that the protein-bound Dnp groups were fully available to the antibody even when the alkyl chain was short. The apparent dissociation constants were significantly greater than that of the interaction between anti-Dnp antibody and the free hapten, 6-(N-Dnp)-aminohexanoate. The antibody fluorescence was quenched efficienty by [dnp-Lys41]ribonuclease A, also with an increased dissociation constant. It could be concluded from the increase in dissociation constant that the Dnp group spent no more than 0.1% of its time in the dissociated state, available to antibody. The second-order rate constants for the association between the Dnp-mercaptablumins and the antibody were determined and were similar in magnitude to those observed in other interactions between protein and anti-protein antibody.
| S-EPMC1164120 | biostudies-other
Project description:The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes.
| S-EPMC1219499 | biostudies-other
Project description:1. Serine and lysine in complicated mixtures, such as protein hydrolysates, are dinitrophenylated quantitatively, isolated chromatographically and determined. 2. The method is accurate, moderately rapid and suitable for a number of estimations in parallel, but uses only normal laboratory apparatus.
| S-EPMC1264830 | biostudies-other