Tobacco protein separation by aqueous two-phase extraction

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.     

Observation of a quadrupole plasmon mode for a colloidal solution of gold nanoprisms

The synthesis and optical properties of single crystalline gold nanoprisms have been investigated. A three-step mediated seed growth process in an aqueous solution generated gold nanoprisms with a relatively homogeneous size distribution. The purity of these nanostructures has allowed us to observe a weak quadrupole resonance in addition to a strong dipole resonance associated with these novel structures. The experimental optical spectra agree with discrete dipole approximation calculations that have been modeled from the dimensions of gold nanoprisms produced in this synthesis.     

The use of novel water-in-oil microemulsions in microemulsion electrokinetic chromatography

We describe the novel use of water-in-oil (W/O) microemulsions to achieve unique separations in microemulsion electrokinetic chromatography (MEEKC). The choice and concentration of the buffer type, surfactant and co-surfactant were all examined and optimized. Separations of a range of neutral and acidic analytes was shown to be markedly different to that obtained by (oil-in-water) O/W MEEKC. Neutral solutes are separated by virtue of their solubility (log P) values in O/W MEEKC with the more water-insoluble solutes migrating last. This separation process does not occur in W/O, as neutral solutes are not separated in order of log P.     

PHOTOSENSITIZATION WITH BACTERIOCHLORINS

Abstract— Biophysical and photobiological properties of a group of bacteriochlorins were compared with efficacy of these products for photodynamic therapy of murine tumors. Predictive factors for selective photosensitization in vivo include affinity binding to lipoproteins greater than albumin, extinction coefficient at the wavelength of irradiation and tumor/skin distribution. Efficacy was correlated with circulating plasma levels of the different sensitizers but not with the photodynamic therapy response in cell culture.     

An ESR study of some group IVB free radical adducts of diethylketomalonate

The formation of the compound RSnX(acac)₂ (acac = 2,4-pentanedionato) by reaction of bis(2,4-pentanedionato)tin(II) on a halide RX with R = CH₃, C₂H₅, C₄H₉, C₆H₅, CH₂I, (C₆H₅)₃SnCH₂, (C₂H₅)₃SnCH₂ and X = I, Br has been studied by polarography. At 25°C, it is in fact an equilibrium whose constant has been measured. The intermediate formation of the ion-pair [RSn(acac)₂⁺X^?] has allowed us to explain the experimental results.     

Automatic absolute determination of total sulfur by combustion and coulometric titration

An automatic method for the absolute determination of total sulfur, which overcomes the tendency of previous methods to give low sulfur recoveries on samples with high oxygen demand, is described. The technique combusts the sample in oxygen and sweeps the combustion gases through a hydrogen peroxide trap to remove the sulfur oxides from the gas stream. The trap contents are volatilized and swept with nitrogen through reduced copper heated to 890 °C. Sulfur oxides are quantitatively converted to sulfur dioxide and titrated coulometrically with iodine. Halogens are held by the copper and do not interfere with the sulfur determination. Analysis time is 12 min and automation of the method allows unattended operation. Sample size is 0.5 to 20 mg. Sulfur levels of 0.05 to 100% can be determined.     

Spectroscopic properties of tyrosyl radicals in dipeptides

Redox-active tyrosine residues play important roles in long-distance electron reactions in enzymes, including prostaglandin H synthase, galactose oxidase, ribonucleotide reductase, and photosystem II. Magnetic resonance and vibrational spectroscopy provide methods with which to study the structures of redox-active amino acids in proteins. In this report, ultraviolet photolysis was used to generate tyrosyl radicals from polycrystalline tyrosinate or dipeptides, and the structure of the radical was investigated with EPR and reaction-induced FT-IR spectroscopy at 77 K. Photolysis at 77 K is expected to generate a neutral tyrosyl radical through oxidation of the aromatic ring. EPR and FT-IR results obtained from (13)C-labeled tyrosine were consistent with that expectation. Surprisingly, labeling of the tyrosyl amino group with (15)N also resulted in isotope-shifted bands in the photolysis spectrum. The force constant of a NH deformation mode increased when the tyrosyl radical was generated. These data suggest an interaction between the pi system of the tyrosyl radical and the amino group. In spectra acquired from the dipeptides, evidence for a sequence-dependent interaction between the tyrosyl radical and the amide bond of the dipeptide was also obtained. We postulate that perturbation of the amino or the amide/imide groups may occur through a spin polarization mechanism, which is indirectly detected as a change in NH force constant. This conclusion is supported by density functional calculations, which suggest a conformationally sensitive delocalization of spin density onto the amino and carboxylate groups of the tyrosyl radical. These experiments provide a step toward a detailed spectral interpretation for protein-based tyrosyl radicals.     

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K _D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC₅₀ of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 μg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).     

Kinetics and mechanism of rhenium-catalyzed O atom transfer from epoxides

O atom transfer from epoxides cis-stilbene oxide and styrene oxide to triphenylphosphine catalyzed by Tp'ReO(3) (Tp' = hydridotris(3,5-dimethylpyrazolyl)borate) is shown to proceed via an unexpectedly complex combination of mechanisms. Reduction of Tp'ReO(3) with PPh(3) in THF is rapid above room temperature to form a highly reactive species suggested to be Tp'ReO(2). Spectroscopic examination and attempts to isolate this by chromatography lead only to Tp'Re(O)(OH)(2) (1); exposure of the crude reduction mixture to ethanol results in formation of Tp'Re(O)(OEt)(OH) (3). Both 1 and 3 are as efficient catalysts for O atom transfer as the unpurified mixture resulting from reaction of PPh(3) with Tp'ReO(3); all three rhenium reactants give the same turnover frequency to within 10% at identical [Re](total) and [epoxide]. The kinetic behavior of the catalytic system (epoxide:Re = 20) is complex; an initial "burst" of alkene production is seen, which quickly tapers off and falls into a pseudo-zero-order reaction. The majority of rhenium is observed to exist as the syn-Tp'Re(O)(diolate) complex, formed by ring expansion of the epoxide. However, cycloreversion of this diolate is incapable of accounting for the observed catalytic turnover frequency. An additional intermediate, a coordinated epoxide, is proposed to form and partition between ring expansion and direct fragmentation to alkene; eventually a steady-state concentration of diolate forms. Competition between direct atom transfer and ring expansion followed by diolate cycloreversion is demonstrated by reaction of 3 with excess cis-stilbene oxide and styrene oxide in the absence of reductant to give a 4:1 mixture of alkene and syn-diolate from cis-stilbene oxide or a 5.5:1 mixture of alkene and syn-diolate from styrene oxide under conditions where diolate cycloreversion is a negligible contributor.     

Studies in bicyclo[3.1.0]hexane methanolysis. Ring opening of activated cyclopropanes under acidic and basic conditions

A bicyclo[3.1.0]hexane, with one cyclopropane carbon flanked by a ketone and an ester or an aldehyde, undergoes methanolysis with cleavage of one of the two activated cyclopropane bonds, depending on the reaction conditions. Acidic conditions yield primarily or exclusively a 4-methoxycyclohexane, while basic conditions yield a 3-methoxymethylcyclopentanone.