Anti-Biotin polyclonal antibody [FITC]

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

The use of reversible addition-fragmentation chain transfer (RAFT)-assisted encapsulating emulsion polymerization (REEP) has been explored to prepare diverse types of colloidal stable core-shell nanostructures. A major field of application of such nanoparticles is in emergent nanomedicines, which require effective biofunctionalization strategies, in which their response to bioanalytes needs to be firstly assessed. Herein, functional core-shell nanostructures were prepared via REEP and click chemistry. Thus, following the REEP strategy, colloidal gold nanoparticles (Au NPs,d= 15 nm) were coated with a poly(ethylene glycol) methyl ether acrylate (PEGA) macroRAFT agent containing an azide (N3) group to affordN3-macroRAFT@Au NPs. Then, chain extension was carried out from the NPs surface via REEP, at 44 degrees C under monomer-starved conditions, to yieldN3-copolymer@Au NPs-core-shell type structures. Biotin was anchored toN3-copolymer@Au NPs via click chemistry using an alkynated biotin to yield biofunctionalized Au nanostructures. The response of the ensuing biotin-copolymer@Au NPs to avidin was followed by visible spectroscopy, and the copolymer-biotin-avidin interaction was further studied using the Langmuir-Blodgett technique. This research demonstrates that REEP is a promising strategy to prepare robust functional core-shell plasmonic nanostructures for bioapplications. Although the presence of azide moieties requires the use of low polymerization temperature, the overall strategy allows the preparation of tailor-made plasmonic nanostructures for applications of biosensors based on responsive polymer shells, such as pH, temperature, and photoluminescence quenching. Moreover, the interaction of biotin with avidin proved to be time dependent.