pH‐Sensitive Mechanical Properties of Elastin‐Based Hydrogels

Ionizable amino acids in protein‐based hydrogels can confer pH‐responsive behavior. Because elastin‐like polypeptides (ELPs) have an established sequence and can crosslink to form hydrogels, they are an ideal system for creating pH‐sensitive materials. This study examines different parameters that might affect pH‐sensitive behavior and characterizes the mechanical and physical properties between pH 3 and 11 of three ELP‐based crosslinked hydrogels. The first finding is that varying the amount of crosslinker affects the overall stiffness and resilience of the hydrogels but does not strongly affect water content, swelling ratio, or pH sensitivity. Second, the choice of two popular tag sequences, which vary in histidine and aspartic acid content, does not have a strong effect on pH‐sensitive properties. Last, selectively blocking lysine and tyrosine residues through acetylation significantly decreases the pH‐sensitive zeta potential. Acetylated hydrogels also demonstrate different behavior at low pH values with reduced swelling, reduced water content, and higher stiffness. Overall, this work demonstrates that ELP hydrogels with ionizable groups are promising materials for environmentally‐responsive applications such as drug delivery, tissue engineering, and microfluidics.     

AFM of adsorbed polyelectrolytes on cellulose I surfaces spin-coated on silicon wafers

Thin layers of cellulose I nanocrystals were spin-coated onto silicon wafers to give a flat model cellulose surface. A mild heat treatment was required to stabilize the cellulose layer. Interactions of this surface with polyelectrolyte layers and multilayers were probed by atomic force microscopy in water and dilute salt solutions. Deflection–distance curves for standard silicon nitride tips were measured for silicon, cellulose-coated silicon, and for polyelectrolytes adsorbed on the cellulose surface. Transfer of polymer to the tip was checked by running deflection–distance curves against clean silicon. Deflection–distance curves were relatively insensitive to adsorbed polyelectrolyte, but salt addition caused transfer of cationic polyelectrolyte to the tip, and swelling of the polyelectrolyte multilayers.     

Matrix-assisted laser desorption/ionisation mass spectrometry for the direct analysis of enzymatically digested kappa- iota- and hybrid iota/nu-carrageenans

Enzymatically digested oligosaccharides of kappa-, iota- and hybrid iota/nu-carrageenans were analysed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the negative-ion mode. nor-Harmane was used as matrix. Depending on the stock concentration and the laser intensity applied, the oligosaccharides exhibited losses of sulphate units (neutralised by the Na+ ion, and thus non-stable), leaving the primary backbone structure in most cases with only the deprotonated sulphate groups (carrying the negative charge, stable). This meant that kappa- and iota-oligosaccharides could not be easily distinguished from one another since they share the same primary backbone structure. However, for the hybrid iota/nu-oligosaccharides the primary backbone structure could be identified since the nu-carrageenan repeating unit differs from that of the kappa/iota-carrageenan unit. For all types of oligosaccharides, the results indicated cleavage of an anhydrogalactose unit from the non-reducing end. Specifically, for the hybrid oligosaccharides of iota/nu-carrageenans, this type of fragmentation means that the nu-carrageenan unit is not positioned on the non-reducing end of the hybrid oligosaccharides. Dehydration reactions, and exchange reactions of Na+ with K+ and Ca2+, were also observed.     

Electrochemical templating of metal nanoparticles and nanowires on single-walled carbon nanotube networks

The use of single-walled carbon nanotube (SWNT) networks as templates for the electrodeposition of metal (Ag and Pt) nanostructures is described. Pristine SWNTs, grown on insulating SiO2 surfaces using catalyzed chemical vapor deposition, served as the working electrode. In the simplest case, electrical contact was made by depositing a gold strip on the SWNT substrate (device 1). Deposition of Ag and Pt over extensive periods (30 s) resulted in a high density of particles on the SWNTs, with almost contiguous nanowire formation from the Au/SWNT boundary moving to isolated nanoparticles at further distances from the contact. For direct electrochemical studies of Ag and Pt nucleation, the assembly was coated in a resist layer and a small window opened up to expose only the electrically connected SWNTs to solution (device 2). In this case, the electrochemical signature in voltammetric and amperometric studies of metal deposition was due solely to processes at the SWNTs. Coupled with high-resolution microscopy measurements (atomic force microscopy and field emission scanning electron microscopy), this approach provided detail on the nucleation and growth mechanisms of Ag and Pt on SWNTs under electrochemical control. In particular, Ag growth was found to be rapid and progressive with an increasing nanoparticle density with time, whereas Pt deposition was characterized by lower nucleation densities and slower growth rates with a tendency for larger particles to be produced over long times.     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

1,2-dioxines containing tethered hydroxyl functionality as convenient precursors for pyran syntheses

A new method for the construction of tetrahydropyrans derived from readily available 1,2-dioxines containing a tethered hydroxyl moiety is described. The reaction proceeds via a base-catalyzed rearrangement of the 1,2-dioxines to either the isomeric cis or trans gamma-hydroxy enones followed by intramolecular oxa-Michael addition of the tethered hydroxyl group.     

Eliminating false positive C4 sugar tests on New Zealand Manuka honey

Carbon isotope analyses (δ¹³C) of some New Zealand Manuka honeys show that they often fail the internationally recognised Association of Official Analytical Chemists sugar test (AOAC method 998.12) which detects added C₄ sugar, although these honeys are from unadulterated sources. Failure of these high value products is detrimental to the New Zealand honey industry, not only in lost export revenue, but also in brand and market reputation damage. The standard AOAC test compares the carbon isotope value of the whole honey and corresponding protein isolated from the same honey. Differences between whole honey and protein δ¹³C values should not be greater than +1.0‰, as it indicates the possibility of adulteration with syrups or sugars from C₄ plants such as high fructose corn syrup or cane sugar. We have determined that during the standard AOAC method, pollen and other insoluble components are isolated with the flocculated protein. These non‐protein components have isotope values which are considerably different from those of the pure protein, and can shift the apparent δ¹³C value of protein further away from the δ¹³C value of the whole honey, giving a false positive result for added C₄ sugar. To eliminate a false positive C₄ sugar test for Manuka honey, prior removal of pollen and other insoluble material from the honey is necessary to ensure that only the pure protein is isolated. This will enable a true comparison between whole honey and protein δ¹³C isotopes. Furthermore, we strongly suggest this modification to the AOAC method be universally adopted for all honey C₄ sugar tests. Copyright © 2010 John Wiley & Sons, Ltd.     

Fragmentation mechanisms of oxidized peptides elucidated by SID, RRKM modeling, and molecular dynamics

The gas-phase fragmentation reactions of singly charged angiotensin II (AngII, DR⁺VYIHPF) and the ozonolysis products AngII+O (DR⁺VY*IHPF), AngII+3O (DR⁺VYIH*PF), and AngII+4O (DR⁺VY*IH*PF) were studied using SID FT-ICR mass spectrometry, RRKM modeling, and molecular dynamics. Oxidation of Tyr (AngII+O) leads to a low-energy charge-remote selective fragmentation channel resulting in the b ₄ +O fragment ion. Modification of His (AngII+3O and AngII+4O) leads to a series of new selective dissociation channels. For AngII+3O and AngII+4O, the formation of [MH+3O] ⁺ −45 and [MH+3O] ⁺ −71 are driven by charge-remote processes while it is suggested that b ₅ and [MH+3O] ⁺ −88 fragments are a result of charge-directed reactions. Energy-resolved SID experiments and RRKM modeling provide threshold energies and activation entropies for the lowest energy fragmentation channel for each of the parent ions. Fragmentation of the ozonolysis products was found to be controlled by entropic effects. Mechanisms are proposed for each of the new dissociation pathways based on the energies and entropies of activation and parent ion conformations sampled using molecular dynamics.