A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer

In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions

Two phase aqueous extraction of whey proteins in a polyethylene glycol - arabinogalactan system

The whey protein separation potential of aqueous two-phase systems of arabinogalactan [AG] (Lonza FiberAidTM) and polyethylene glycol [PEG], buffered with 10 mmol/g phosphate or citrate buffer, was studied. 100 mmol/g potassium chloride [KCl] was added as required. Previously-published phase equilibrium results were verified and the absorbance of whey protein isolate [WPI] in an AG-PEG solution was measured. The effect of pH, KCl concentration, initial WPI concentrations and upper to lower phase mass ratios on whey partitioning was studied. The best separation system contained 17.20% (w/w) AG, 7.20% (w/w) PEG, 10 mmol citrate buffer (pH 5.4) and 100 mmol KCl per gram of total system. The upper to lower phase mass and volume ratios were 1:1 and 16:11 respectively. Approximately 12 mg (mainly α-lactalbumin) of the 20 mg WPI added partitioned into the AG-rich upper phase. This system has potential to reduce chromatographic requirements in large scale separation of protein mixtures

Improved pH buffering agent for sodium hypochlorite

Sodium citrate/citric acid was found to be an effective buffer for pH control when used with sodium hypochlorite. The mixture does not corrode aluminum. The buffer appears to form a type of conversion coating that may provide corrosion-resistant properties to aluminum in other applications

Postharvest characteristics of cut flowers of selected members of the family Myrtaceae : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy at Massey University, New Zealand

Stages of floral development were described for Eucalyptus ficifolia and Metrosideros collina 'Tahiti' flowers (Myrtaceae) attached and detached from plants. Vase solution treatments were applied to promote bud opening of cut flowers and to prevent postharvest stamen wilting and abscission in both species. Water uptake and mass of harvested flowers in both species declined rapidly when the pedicels were placed in water (control). Some flower buds did not open after harvest. The decline in water uptake and flower mass was greatly reduced by a vase solution treatment containing 2% sucrose, and 200 ppm hydroxyquinoline citrate (HQC) adjusted to pH 4 using citrate buffer. Vase solutions containing higher sucrose concentrations (more than 6%) and of greater acidity (pH<4) were not beneficial for vase life of both species. Cut flowers of both species held in the standard solution (2% sucrose, 200 ppm HQC adjusted to pH 4 using citrate buffer) were treated with ethephon (0-10,000 ppm) following pre-treatment with silver thiosulphate (STS) (0-2.0 mM). Ethephon treatments significantly induced stamen wilting, but had no effect on stamen or petal abscission in both species. Pre-treatment with 2 mM STS had no effect on the rate of stamen wilting, but significantly reduced stamen or petal abscission in both species. Cut flowers of M. collina 'Tahiti' held in the standard solution were treated with ethylene (0-5 ppm). Exogenous ethylene significantly promoted abscission of stamens and petals in M. collina 'Tahiti'. Treatment with 0.5 and 5 ppm ethylene also induced flower abscission. Ethylene emanation from untreated cut flowers from plants grown in two environments (greenhouse and outside) was also measured. Untreated cut flowers harvested from plants grown outside produced more endogenous ethylene than those from plants grown in the greenhouse. The abscission of M. collina 'Tahiti' probably results from a relatively high sensitivity to ethylene

Esterase Activity from the Germinated Jatropha curcas Seeds in Different Extraction buffers.

The buffer solution plays a major role in protein stability and activity, thereby making the selection of a buffer to achievemaximum activity for a protein will be a formidable challenge. The present work constitutes an extension of this investigation to esterases from germinated Jatrophacurcas seeds. The 0.1 M NaCl solution, 0.1 M phosphate buffer pH7.0, 0.1 M citrate buffer pH 5.0 and 0.1 M Tris-HCl buffer pH 8.5, 0.1 M NaOH and distill water were used to extract protein from germinated Jatrophacurcas seeds. The esterase activity and specific activity for NaCl solution, phosphatebuffer, citrate buffer, Tris-HCl buffer, NaOH and Distilled water was 9.07, 8.6, 8.2, 6.46, 0.07 and 4.98 μmoles/min/gm and 0.09258, 0.0905, 0.088, 0.0715 0.0003 and 0.081 IU/mg, respectively.The Native-PAGE analysis showed the esterase enzyme activity in different extraction buffer.Among 13 esterase bands, 8 esterolytic bands were major bands (band no 1,3, 6, 7, 8, 11, 12 and 13)and remaining were minor bands.The amount of proteins and esterase activity were found to bethe highest when extracted with 0.1 M NaCl solution

Development of Mucoadhesive Gel Microbicide to Target Mucosal HIV Reservoirs

The wide use of microbicide is mainly depends on its effectiveness, less frequent application, ready availability and most importantly cost. The aim of this work was to develop affordable microbicide mucoadhesive gel formulation of synthetic anti HIV drug, stavudine and to characterise it in terms of its physical properties, mucoadhesiveness and spreadability. The purpose of the present study was also to compare different dissolution media used for in vitro release of vaginal dosage form. The gels were tested for antimicrobial, spermicidal and anti-HIV activity. Gels prepared using Carbopols and Polycarbophil were transparent and homogenous and had excellent mucoadhesion index - and showed fast drug release profile. Gels showed very good antimicrobial action against pathological microorganism

Trichoderma reesei derived cellulase activity in three N,N-dimethylethanolammonium akylcarboxylate ionic liquids

The activity and denaturation extent of cellulase from Trichoderma reesei (E.C. # 3.2.1.4) was investigated in three representative N,N-dimethylethanolammonium akylcarboxylate ionic liquids. Significant cellulase activity and absence of enzyme unfolding was found in all concentrations of N,N-dimethylethanolammonium acetate (DMEAA), including the pure liquid. Activities in 20% and 40% (v/v) solutions of DMEAA were equal to citrate buffer controls. Lower enzymatic activities and denaturation were observed in solutions of the corresponding formate and octanoate ionic liquids, although cellulose hydrolysis still proceeded at a substantial rate. The results provide the first proof-of-principle that cellulose can be enzymatically hydrolyzed in the presence of high ionic liquid concentrations

Short-Chained Oligo(Ethylene Oxide)-Functionalized Gold Nanoparticles: Realization Of Significant Protein Resistance

Protein corona formed on nanomaterial surfaces play an important role in the bioavailability and cellular uptake of nanomaterials. Modification of surfaces with oligoethylene glycols (OEG) are a common way to improve the resistivity of nanomaterials to protein adsorption. Short-chain ethylene oxide (EO) oligomers have been shown to improve the protein resistance of planar Au surfaces. We describe the application of these EO oligomers for improved protein resistance of 30 nm spherical gold nanoparticles (AuNPs). Functionalized AuNPs were characterized using UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Capillary electrophoresis (CE) was used for separation and quantitation of AuNPs and AuNP-protein mixtures. Specifically, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was employed for the determination of equilibrium and rate constants for binding between citrate-stabilized AuNPs and two model proteins, lysozyme and fibrinogen. Semi-quantitative CE analysis was carried out for mixtures of EO-functionalized AuNPs and proteins, and results demonstrated a 2.5-fold to 10-fold increase in protein binding resistance to lysozyme depending on the AuNP surface functionalization and a 15-fold increase in protein binding resistance to fibrinogen for both EO oligomers examined in this study

Conserved Residues R420 and Q428 in a Cytoplasmic Loop of the Citrate/Malate Transporter CimH of Bacillus subtilis Are Accessible from the External Face of the Membrane

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET, which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.