Co-extraction during reactive extraction of phenylalanine using Aliquat 336: interfacial mass transfer

Reactive liquid-liquid extraction can be used to separate hydrophilic fermentation products that would not otherwise partition into nonpolar solvents. However, during extraction of the target solute other compounds present in the extraction medium will also react with the ion exchange reagent and are thus co-extracted. In this study the effect of co-extraction on the interfacial flux of the target solute phenylalanine has been investigated for reactive extraction using Aliquat 336. The effect of co-extracting compounds has been included in a new interfacial flux balance, and experimental results reveal that the interfacial concentrations are equal to the final equilibrium conditions of the system. Using this information a simple mass transfer model has been developed from which film mass transfer coefficients may be determined. Co-extraction of other compounds present in the feed was found to reduce the interfacial flux of the target solute by reducing the driving force. Co-extraction did not affect the value of the film mass transfer coefficient, and therefore, co-extraction does not effect the transport properties of the solute to the interface. Extraction from a multicomponent fermentation broth resulted in a reduced flux, which arises from a reduction in the driving force caused by high levels of co-extraction. Furthermore, the flux was also reduced as the result of a mass transfer resistance caused by soluble surface-active compounds present in the fermentation broth adsorbing to the interface. The biomass associated with the fermentation broth was also found to reduce the solute flux, and it is believed that this is due to blockage of the interfacial area.     

Coextraction during reactive extraction of phenylalanine using Aliquat 336: modeling extraction equilibrium

Reactive liquid-liquid extraction can be used to recover hydrophilic fermentation products that would not otherwise partition into nonpolar solvents through an ion-exchange reaction at the two-phase interface. However, the ion-exchange reagent may not be specific to the solute of interest and other compounds present may also be extracted. In this study, the effect on solute extraction of other compounds present in the extraction medium was investigated for phenylalanine extraction using Aliquat 336. The extent of extraction at equilibrium was modeled using the equilibrium constants for the reactions present in the process. The interaction of different species within a multicomponent medium was examined using the model and experimental results. It was found that the extent of extraction and coextraction is controlled by the thermodynamics of each extraction reaction and, due to the formation of a common product, the interaction between each of the reactions. The main competition to reactive extraction will come from hydrophobic anionic compounds that will be present in fermentation broth.     

Investigations of biocompatible systems for reactive extraction of propionic acid using aminic extractants (TOA and Aliquat 336)

This paper presents the reactive extraction of propionic acid from aqueous solution by amine based extractants such as tri-n-octylamine and Aliquat 336, dissolved in a mixture of n-dodecane and 1-decanol. Equilibrium experiments were carried out to investigate the effects of various parameters such as modifier (1-decanol) concentration, extractant type, extractant composition, diluent composition, and initial acid concentration on the extraction efficiency. The extraction efficiency was found to be increased with an increase in modifier composition and extractant composition, and decreased with increases in initial acid concentration. Different biocompatible extractant/diluent systems such as (1) 20% TOA, 20% 1-decanol and 60% n-dodecane, (2) 20% TOA, 30% 1-decanol and 50% n-dodecane, (3) 30% TOA, 20% 1-decanol and 50% n-dodecane and (4) 25% Aliquat 336, 25% 1-decanol and 50% n-dodecane are developed and used in this study. A mathematical model based on mass action law and a population-based search algorithm (differential evolution, DE) is proposed, and is used to estimate the extraction equilibrium constant (K _E) and stoichiometry of reactive extraction. Individual equilibrium constants for the simultaneous formation of (1:1) and (2:1) acid:amine complexes are also determined. The extraction system comprised of 20% TOA, 30% 1-decanol, and 50% n-dodecane was found to be the best among the four biocompatible extractant/diluent systems studied. The loading ratios found in the range of 0.113 ～ 1.05 indicated the simultaneous formation of 1:1 and 2:1 complexes between acid and TOA.     

Factors influencing the potential use of Aliquat 336 for the in situ extraction of carboxylic acids from cultures of Pseudomonas putida

The use of extraction techniques to alleviate product inhibition in bioprocesses is one of a number of potential separation methods. However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous phase at saturation levels. The quaternary amine Aliquat 336 (trioctyl/decylmethylammonium entity), dissolved in octan-1-ol showed no inhibition on the growth of Pseudomonas putida, at least with respect to molecular toxicity. Nevertheless, it is important to point out two main effects of Aliquat 336 associated with its ion exchange properties. It is able (1) to complex hydroxyl ions and therefore drastically lower the pH of the broth and (2) release its counter ion through these exchanges. Therefore, a strict control of the pH of the cultivation must be conducted, with the constraint that Ps. putida has an optimal pH growth of 7.4-7.5. The pH range tolerated by this strain is, however, between 5.0 and 9.0. In addition, the counter ion of Aliquat 336 needs to be carefully chosen and HSO4- should be preferred to Cl-.     

Purification of industrial alphaamylase by reversed micellar extraction

The purification of industrial alpha-amylase by liquid-liquid extraction with Aliquat 336 reversed micellar solution as the extractant was studied. Seven kinds of Aliquat 336 reversed micellar solution, formed by using seven kinds of straight chain alkyl alcohols as cosolvent, have been utilized to extract industrial a-amylase. It was found that these seven kinds of reversed micellar solution can all achieve a high protein transfer efficiency in the forward extraction process. After a full forward and backward extraction cycle, however, only the reversed micelles with n-butanol as the cosolvent was found to be able to maintain the activity of alpha-amylase in the stripping solution. By using the reversed micelles of Aliquat 336/isooctane/1% (v/v) n-butanol to perform a full extraction cycle, it was found that 85% of the total activity of alpha-amylase in the industrial a-amylase could be recovered at the end of an extraction cycle and the specific activity of alpha-amylase could be concentrated about 1.5-fold; meanwhile, most of the neutral protease in the industrial a-amylase could be removed. The separation factor of alpha-amylase to neutral protease at the end of an extraction cycle can reach about 10. (c) 1995 John Wiley & Sons, Inc.     

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.     

Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of organic phase, fermentation media, and operating conditions on lactic Acid extraction

Lactic acid has extensive uses in the food, pharmaceutical, cosmetic and chemical industry. Lately, its use in producing biodegradable polymeric materials (polylactate) makes the production of lactic acid from fermentation broths very important. The major part of the production cost accounts for the cost of separation from very dilute reaction media where productivity is low as a result of the inhibitory nature of lactic acid. The current method of extraction/separation is both expensive and unsustainable. Therefore, there is great scope for development of alternative technology that will offer efficiency, economic, and environmental benefits. One of the promising technologies for recovery of lactic acid from fermentation broth is reactive liquid-liquid extraction. In this paper the extraction and recovery of lactic acid based on reactive processes is examined and the performance of a hydrophobic microporous hollow-fiber membrane module (HFMM) is evaluated. First, equilibrium experiments were conducted using organic solutions consisting of Aliquat 336/trioctylamine (as a carrier) and tri-butyl phosphate (TBP)/sunflower oil (as a solvent) The values of the distribution coefficient were obtained as a function of feed pH, composition of the organic phase (ratio of carrier to solvent), and temperature (range 8-40 degrees C). The optimum extraction was obtained with the organic phase consisting of a mixture of 15 wt % tri-octylamine (TOA) and 15% Aliquat 336 and 70% solvent. The organic phase with TBP performed best but is less suitable because of its damaging properties (toxicity and environmental impact) and cost. Sunflower oil, which performed moderately, can be regarded as a better option as it has many desirable characteristics (nontoxic, environment- and operator-friendly) and it costs much less. The percentage extraction was approximately 33% at pH 6 and at room temperature (can be enhanced by operating at higher temperatures) at a feed flow rate of 15-20 L/h. These results suggest that the hollow-fiber membrane process yields good percentage extraction at the fermentation conditions and its in situ application could improve the process productivity by suppressing the inhibitory effect of lactic acid.     

Reactive extraction of lactic acid using alamine 336 in MIBK: equilibria and kinetics

Lactic acid is an important commercial product and extracting it out of aqueous solution is a growing requirement in fermentation based industries and recovery from waste streams. The design of an amine extraction process requires (i) equilibrium and (ii) kinetic data for the acid-amine (solvent) system used. Equilibria for lactic acid extraction by alamine 336 in methyl-iso-butyl-ketone (MIBK) as a diluent have been determined. The extent to which the organic phase (amine +MIBK) may be loaded with lactic acid is expressed as a loading ratio, z=[HL](o)/[B](i,o). Calculations based on the stoichiometry of the reactive extraction and the equilibria involved indicated that more lactic acid is transferred to the organic phase than would be expected from the (1:1) stoichiometry of the reaction. The extraction equilibrium was interpreted as a result of consecutive formation of two acid-amine species with stoichiometries of 1:1 and 2:1. Equilibrium complexation constant for (1:1) and (2:1) has been estimated. Kinetics of extraction of lactic acid by alamine 336 in MIBK has also been determined. In a first study of its kind, the theory of extraction accompanied by a chemical reaction has been used to obtain the kinetics of extraction of lactic acid by alamine 336 in MIBK. The reaction between lactic acid and alamine 336 in MIBK in a stirred cell falls in Regime 3, extraction accompanied by a fast chemical reaction occurring in the diffusion film. The reaction has been found to be zero order in alamine 336 and first order in lactic acid with a rate constant of 1.38 s(-1). These data will be useful in the design of extraction processes.     

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 4. The torsion angle distribution about the C3''-O3'' bond in DNA constituents.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.     

Two-state equilibria of myosin subfragment 1 and its complexes with ADP and actin

We previously reported that the nucleotide complex of myosin subfragment 1, S1.epsilon ADP, exists in two states on the basis of the temperature dependence of the fluorescence decay of bound 1,N6-ethenoadenosine diphosphate (epsilon ADP) [Aguirre, R., Lin. S.-H., Gonsoulin, F., Wang, C.-K., & Cheung, H.C. (1989) Biochemistry 28, 799-809]. We have extended the previous study of the equilibrium between the two states, S1L.ADP in equilibrium S1H.ADP, by using a fluorescently labeled myosin S1 (S1-AF). In S1 alkylated with IAF [5-(iodoacetamido)fluorescein], the decay of the label emission was biexponential both in the presence and absence of ADP and/or actin. In the presence of ADP, the two decay times were 4.30 (alpha 1 = 0.55) and 0.80 ns (alpha 2 = 0.45) at 12.4 degrees C, in a medium containing 60 mM KCl, 30 mM TES (pH 7.5), and 2 mM MgCl2. The steady-state fluorescence intensities of S1-AF, (S1-AF).ADP, acto.(S1-AF), and acto.(S1-AF).ADP were dependent on temperature over the range of 5-30 degrees C. By combining lifetime and steady-state intensity data, we obtained for the two-state transition (S1-AF)L.ADP in equilibrium (S1-AF)H.ADP the following parameters: delta H degrees = 16.1 kcal/mol (67.3 kJ/mol) and delta S degrees = 55.8 cal/(deg.mol) [233.5 J/(deg.mol)], in agreement with previous results obtained with epsilon ADP. The delta H degrees values for the two-state transition of S1-AF, acto.(S1-AF), and acto.(S1-AF).ADP are 13.0, 21.6, and 5.2 kcal/mol, respectively. The corresponding delta S degrees values are 46.9, 79.5, and 17.4 cal/(deg.mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effect of a Quaternary Amine (Aliquat 336) on Growth and Photosynthesis of the Green Alga Chlorella emersonii, and the Effect on Photosynthesis in Isolated Spinach Chloroplasts

A quaternary amine, Aliquat 336, inhibits the growth of the green alga Chlorella emersonii, ¹⁴C-fixation of the alga is also inhibited. The effect and the site of action of the compound was studied by using isolated spinach chloroplasts. The carbon dioxide dependent oxygen evolution of the chloroplasts is inhibited directly upon the addition of the amine and the oxygen evolution is replaced by an oxygen uptake. By investigating some electron transport reactions in the chloroplasts we were able to show that Aliquat 336 affects the electron transport on the level of photophosphorylation. The results from the in vivo and the in vitro experiments thus show that the quaternary amine affects the photosynthetic process. Aliquat 336 is a solvent extractant used in several industrial processes for extraction of metals from aqueous solutions. Aliquat 336 could be considered a presumptive water pollutant as the compound could enter a recipient water body and thus affect photosynthesis.     

Improving the membrane permeability of sialic acid derivatives

The potential of boronic acids to improve the bioavailability of carbohydrate derived drugs was investigated through the study of the transport of four sialic acid derivatives through a lipophilic supported liquid membrane at departure phase pH's of 7.4, 8.5 and 10.0. It was found that facilitated transport did occur in most cases, but interestingly, and in contrast to that observed with monosaccharides such as d-fructose, the lipophilic ammonium salt, Aliquat 336, promoted fluxes than those of the boronic acid. The triol side chain of the sialic acid derivatives, combined with the amide at C5, appears to represent a previously unrecognised chloride binding domain which promotes extraction of these compounds into membranes containing Aliquat 336, leading to fluxes greater than those produced by boronic acids.     

The kinetics of chain exchange in two-chain coiled coils: alpha alpha- and beta beta-tropomyosin.

Measurements are presented on the time course of chain exchange among two-chain alpha-helical coiled coils of rabbit tropomyosin. All experiments are in a regime (temperature, protein concentration) in which coiled-coil dimers are the predominant species. Self-exchange in alpha alpha-tropomyosin was investigated by mixing alpha alpha species with alpha* alpha*, the asterisk designating an alpha-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. The overall process alpha alpha + alpha* alpha* in equilibrium with 2 alpha alpha* is followed by measurement of the fraction (h) of alpha alpha* species as a function of time. Similarly, self-exchange in beta beta-tropomyosin is examined by measurements of the overall process: beta beta + beta* beta* in equilibrium with 2 beta beta*, in which beta* signifies a beta-chain blocked at both sulfhydryls (C36 and C190). The observed time course for both chains is well fit by the first-order equation: h (t) = h (infinity) (1-e-k1t), with h (infinity) congruent to 0.5. This long-time limit is as expected for self-exchange, and agrees with experiments that attain equilibrium after slow cooling of thermally dissociated and unfolded chains. The simplest consonant mechanism is chain exchange by rate-limiting dissociation of dimers followed by random reassociation. Kinetic analysis shows k1 to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for beta beta species is about an order of magnitude greater than for alpha alpha. In both, the activation enthalpy and entropy are very large, suggesting that activation to an extensively (greater than 50%) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: alpha alpha + beta* beta* in equilibrium with 2 alpha beta* and alpha* alpha* + beta beta in equilibrium with 2 alpha* beta. Results are independent of which chain is blocked. Again h (infinity) congruent to 0.5, in agreement with equilibrium experiments, and the time course is first order. The rate constants and activation parameters are intermediate between those for self-exchange.     

The presence in a mouse T cell line of a 97-kDa protein kinase C (PKC) with characteristics similar to known members of the novel PKC subgroup and its possible role in lymphocyte gene expression.

The receptor for tumor-promoting phorbol esters has been shown to be the Ca+2/phospholipid dependent enzyme protein kinase C (PKC). There are two major groups of PKC, the conventional PKC isotypes alpha, beta I, beta II, gamma) and the novel Ca+2-independent PKC (delta, epsilon, zeta, eta). Phorbol esters previously have been demonstrated to increase human IFN-gamma gene expression after treatment of a murine T cell line (Cl 9) that has been transfected with human IFN-gamma genomic DNA. In contrast, treatment with Ca+2 ionophore alone or in combination with phorbol ester did not enhance IFN-gamma production in a synergistic manner above the level obtained with phorbol ester treatment alone. To determine whether the lack of effect of Ca+2 ionophore is due to a defect in PKC, we compared the level of PKC autophosphorylation in the mouse T cell line (Cl 9), a mouse epidermal cell line (JB6), and purified rat brain PKC by in vitro kinase assays. The results demonstrate that instead of the expected 80-kDa autophosphorylated PKC band seen in purified rat brain PKC or mouse JB6 cell lysates, only a novel 97-kDa Ca+2-independent phosphoprotein was observed in Cl 9 cells. To ascertain if there was any nucleic acid sequence similarity to PKC epsilon, we hybridized Cl 9 poly(A+) RNA with a cloned fragment of the PKC epsilon gene and observed two hybridizing RNA bands (4.4 and 4.0 kb). Our results suggest that the 97-kDa phosphoprotein is similar to, but not identical with, PKC epsilon and is the major PKC expressed in the Cl 9 murine T cell line. These data suggested that the 97-kDa PKC may be responsible for the induction of both the transfected human IFN-gamma gene and the endogenous murine IL-2R alpha-chain.     

Extraction of organic acids from kiwifruit juice using a supported liquid membrane process

In order to extract or remove organic acids from kiwifruit juice, we evaluated their separation and transport rates through supported liquid membranes (SLMs). The liquid membrane consisted of an organic solution composed of a carrier (Aliquat 336/Alamine 336) and a linear alcohol (oleyl alcohol) and was loaded on a microporous polypropylene support (commercial grade Celgard 2500/2400). These SLMs were evaluated (i) in a batch cell to determine the permeability and (ii) in a continuous spiral membrane module to study the effects of various process parameters – flow of feed and strip solutions, membrane composition, recycling mode of operation and kiwifruit juice at natural pH. It was observed that there exists an optimum for each system: pH?2.5–?3.0 for Alamine 336/oleyl alcohol and pH?4.5 for Aliquat 336/oleyl alcohol. At this pH?the flux rates of citric acid and malic acid was greater (6–8 times) than that of quinic acid. The flux rates decreased (greatly for citric acid) with the flow rate of feed and strip solutions and increased (considerably for citric acid) with the SLM composition . The recycling of feed and strip solutions significantly improved the removal efficiency. The SLM system retained its performance over a period of a few days. The SLM process allowed extraction of the above three organic acids (ascorbic acid was removed in trace amounts) from kiwifruit juice at a rate of a few percent (5%) in a single-pass processing.     

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.     

Persistence of Tandem Arrays: Implications for Satellite and Simple-Sequence Dnas

Recombination processes acting on tandem arrays are suggested here to have probable intrinsic biases, producing an expected net decrease in array size following each event, in contrast to previous models which assume no net change in array size. We examine the implications of this by modeling copy number dynamics in a tandem array under the joint interactions of sister-strand unequal crossing over (rate gamma per generation per copy) and intrastrand recombination resulting in deletion (rate epsilon per generation per copy). Assuming no gene amplification or selection, the expected mean persistence time of an array starting with z excess copies (i.e., array size z + 1) is z(1 + gamma/epsilon) recombinational events. Nontrivial equilibrium distributions of array sizes exist when gene amplification or certain forms of selection are considered. We characterize the equilibrium distribution for both a simple model of gene amplification and under the assumption that selection imposes a minimal array size, n. For the latter case, n + 1/alpha is an upper bound for mean array size under fairly general conditions, where alpha(= 2 epsilon/gamma) is the scaled deletion rate. Further, the distribution of excess copies over n is bounded above by a geometric distribution with parameter alpha/(1 + alpha). Tandem arrays are unlikely to be greatly expanded by unequal crossing over unless alpha much less than 1, implying that other mechanisms, such as gene amplification, are likely important in the evolution of large arrays. Thus unequal crossing over, by itself, is likely insufficient to account for satellite DNA.