Peptide hydrophobicity and partitioning in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems.

Several amino acids and peptides were partitioned in poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase systems. The partition coefficients measured for amino acids and peptides were proportional to the difference in PEG concentration between the phases. The partitioning data were used to calculate the relative hydrophobicities of individual amino acids, which were then used to estimate the hydrophobicities of peptides. The partition coefficients of several dipeptides were predicted from these estimated hydrophobicities. A series of peptide fragments that compose the pentapeptide leucine enkephalin was also partitioned in the PEG/MgSO4 system. Again, the partitioning depended upon the hydrophobicities of the individual exposed amino acids.     

Feasible boundaries of aqueous two-phase systems with NH(3) and CO(2) as recyclable volatile salts

Aqueous two-phase systems (ATPSs) have great potential for use in the downstream processing of fermentation products. A major drawback of these systems, limiting application in industrial practice up till now, is the consumption of large amounts of auxiliary materials such as polymers and salts. Making use of alternative auxiliaries can diminish this relatively large discharge. A possible approach is to make use of volatile salts induced by combinations of ammonia and carbon dioxide that can be recycled to the extraction system. As part of an ongoing research effort on ATPSs with volatile salts, this work aims at getting more information on the system boundaries or operating conditions of these systems in terms of phase behavior. The results show that the NH(3)/CO(2) ratio is an important parameter that has a large influence on the system boundaries. Both for systems with PEG 2000 and PEG 4000, this ratio has to be larger than about 1.75 to make a liquid-liquid phase separation possible. The most optimal ratio seems to be 2.0 for reasons of solution composition and absence of solid salt.     

Effects of low urea concentrations on protein-water interactions

Solvent properties of aqueous media (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) were measured in the coexisting phases of Dextran–PEG aqueous two-phase systems (ATPSs) containing .5 and 2.0 M urea. The differences between the electrostatic and hydrophobic properties of the phases in the ATPSs were quantified by analysis of partitioning of the homologous series of sodium salts of dinitrophenylated amino acids with aliphatic alkyl side chains. Furthermore, partitioning of eleven different proteins in the ATPSs was studied. The analysis of protein partition behavior in a set of ATPSs with protective osmolytes (sorbitol, sucrose, trehalose, and TMAO) at the concentration of .5 M, in osmolyte-free ATPS, and in ATPSs with .5 or 2.0 M urea in terms of the solvent properties of the phases was performed. The results show unambiguously that even at the urea concentration of .5 M, this denaturant affects partitioning of all proteins (except concanavalin A) through direct urea–protein interactions and via its effect on the solvent properties of the media. The direct urea–protein interactions seem to prevail over the urea effects on the solvent properties of water at the concentration of .5 M urea and appear to be completely dominant at 2.0 M urea concentration.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Fatty acid and alcohol partitioning with intestinal brush border and erythrocyte membranes

Summary Relative partition coefficients of fatty acids and alcohols between aqueous buffers and biological membranes have been determined from the linear relationship between isotope content of sedimented membranes and aqueous concentration. This technique allows study of highly lipid soluble compounds such as long-chain saturated fatty acids. Rat intestinal brush border membranes and erythrocyte ghost membranes were studied by using homologous series of saturated fatty acids mono-unsaturated fatty acids and 10, 12, and 14 carbon normal alcohols. The influence of chain length on partitioning was similar in the three series with an incremental, free energy of –820 cal/mole per methylene group in brush borders for the saturated fatty acids. Incremental enthalpy and entropy were –1331 cal/mole and –1.64 cal/mole,^oK respectively. Decrease in the partition coefficient due to the double bond (monounsaturated relative to saturated) had an incremental free energy of +1178 cal/mole, incremental enthalpy of –3453 cal/mole, and incremental entropy of –7.34 cal/mole,^oK, while substitution of the hydroxyl for the ionized carboxyl group (pH 7.4) increased the partition coefficient by 72-fold. From these data it must be concluded that the lipid phase of the membrane bilayer is extremely hydrophobic, similar to heptane or polyethylene in polarity.     

Genetic code correlations: Differential rates of non-enzymatic activation of hydrophobic amino acids by ATP

The relative rates of non-enzymatic activation of several hydrophobic amino acids by ATP have been found to bear an interesting relationship to the ordering of these amino acids in the genetic anticode. All of these hydrophobic amino acids (phe, leu, val, ile and met) have adenylic acid, the most hydrophobic nucleotide, as the central and most important member of their anticodons, and the ordering of their relative rates of nonenzymatic activation by ATP has been found not to correlate with the ordering of the hydrophobicities of the amino acids themselves, but rather with the ordering of the average estimates of the hydrophobicities of their respective anticodonic dinucleotides. These data suggest that the genetic code is based not just on hydrophobic relationships or affinities between amino acids and nucleotides, but perhaps more importantly, on the total reaction chemistry between amino acids and nucleotides.     

Partitioning of amino acids in the aqueous biphasic system containing the water‐miscible ionic liquid 1‐butyl‐3‐methylimidazolium bromide and the water‐structuring salt potassium citrate

In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio‐molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)—a new class of non‐volatile alternative solvents. ABS composed of the hydrophilic IL {1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br)} and potassium citrate—which is easily degraded—represents a clean media to green separation of bio‐molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL ‐based ABS, the distribution of five model amino acids (L ‐tryptophan, L ‐phenylalanine, L ‐tyrosine, L ‐leucine, and L ‐valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting‐out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL ‐based ABS, a model was established to describe the partition coefficient of three model amino acids, L ‐tryptophan, L ‐phenylalanine, and L ‐valine, and employed to predict the partition coefficient of two other model amino acids, L ‐tyrosine and L ‐leucine. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Mechanisms of amino acid partitioning in cationic reversed micelles

The aim of this work is to discuss the mechanisms involved in amino acidsolubilization in cationic reversed micelles. A simple mechanism was assumedin which the amino acid solubilization is mediated by an ion-exchangeprocess between the amino acid and the surfactant counter ion neglecting theeffect of the reversed micellar structure. Based on this mechanism a simplemodel to predict equilibrium was developed and applied to the solubilizationof amino acids with different structures. It was found that solubilizationof hydrophilic and slightly hydrophobic amino acids can be described by anion-exchange mechanism and the amino acid equilibrium concentration can bedetermined for different experimental conditions using this model. However,solubilization of hydrophobic amino acids can not be described by a simpleion-exchange model. In this case hydrophobic contributions play an importantrole in amino acid solubilization and must be considered in the overallsolubilization process. This hydrophobic contribution was evaluated bydetermination of an interfacial partition coefficient. The overall aminoacid extraction was determined using distribution coefficients of all theamino acid forms and considering their dependence on ionic strength.     

Purification and aqueous two-phase partitioning properties of recombinant Vitreoscilla hemoglobin.

Soluble recombinant Vitreoscilla hemoglobin was purified from E. coli lysate by sequential two-phase extraction techniques. Extraction of lysate containing VHb in PEG/dextran gave a 3.6-fold increase in VHb purity in the PEG-rich phase via a size exclusion mechanism. Further extraction of the recovered PEG phase in PEG/sodium sulfate gave an additional 2.0-fold increase in purity in the PEG-rich phase due to an electrostatic mechanism. Final extraction of the PEG phase in PEG/magnesium sulfate gave an additional 1.3-fold increase in VHb purity in the magnesium sulfate-rich phase. The final yield from the extractive purification was 47% with purity of VHb estimated to be greater than 95%. Yields from the sulfate salt extractions are essentially quantitative due to the extreme partitioning behavior of VHb in these systems. VHb partition coefficients as large as 46 in PEG/sodium sulfate and as small as 0.06 in PEG/magnesium sulfate were observed. Similar small partition coefficients were obtained with PEG/manganese sulfate extractions. This dramatic effect of divalent cation content on the partition coefficient of VHb in PEG/sulfate salt systems was investigated by pH and magnesium ion titration experiments. Results show the effect to be largest and nearly constant for pH values greater than 6.0 and diminished at lower pH values. A model based on magnesium ion binding to negatively charged amino acids is shown to correlate with the data well. Based on model formulation and the partitioning behavior of contaminant proteins, the observed effect is expected to be applicable to other proteins.     

Xylanase mass transfer studies in aqueous two-phase systems using spray and sieve plate columns

Aqueous two-phase systems (ATPSs) have long been used for biomolecule partitioning; these systems offer the possibility of using continuous or semicontinuous extraction processes. They require relatively simple equipment like spray or sieve plate columns that can be adapted for use in ATPSs. The aim of this work was to study the semicontinuous extraction of a model enzyme, xylanase, in spray and sieve plate columns, since, unlike centrifugal contactors, the cost of construction and maintenance of this equipment is low and it is easy to operate. For the spray column, the dispersed phase hold-up and overall mass transfer coefficients K(D) a were evaluated for different column heights and for different superficial velocities of the dispersed phase (light phase). Results indicated that an increase in superficial velocity in the range of 0-0.18 mm/s of the dispersed phase had a positive effect on K(D) a and on hold-up in all column heights studied, 75, 161 and 246 mm. For the same superficial velocity of the dispersed phase, the larger the hold-up was, the shorter the column. For the sieve plate column, the effects of the superficial velocity of the dispersed phase and the number of plates were also studied. Results showed that the K(D) a and hold-up increased with an increase in both parameters. The selectivity of separation of xylanase and BSA (model contaminant) was very high, since 60% of the enzyme was extracted in the light phase, whereas no significant amount of BSA was extracted. The possibility of using the sieve plate column in continuous operation for enzyme extraction was studied because previous work had only addressed the semicontinuous extraction of enzyme. The residence time distribution of the PEG phase using different superficial velocities of the salt phase was studied in continuous operation. The time required to reach the steady state was 40 min, and 70% of the xylanase was recovered. It was found that the Modified Power Spline software was well adjusted to the experimental results.     

The relationship between hydrophobicity and interfacial tension of proteins.

Charge-free hydrophobic gels of Hjerten et al. (Hjerten, S., Rosengren, J. and Pahlman, S. (1974) J. Chromatogr. 101, 281--288) were used for hydrophobic affinity chromatography. The effective hydrophobicity of proteins was expressed as their retention volumes from columns of butylepoxy- and hexylepoxy-Sepharose 4B. The effective hydrophobicity was also estimated by a partition method of Shanbhag and Axelsson ((1975) Eur. J. Biochem. 60, 17--22) from the partition coefficients of proteins between two phases, poly (ethylene glycol) and dextran. The former contained a hydrophobic ligand, palmitate. A close correlation was observed between the hydrophobicities determined by the two methods. However, no significant relationship was observed between these effective hydrophobicities and the average hydrophobicity of Bigelow ((1967) J. Theoret. Biol. 16, 187--211) that was calculated from the total amino acid composition of each protein. The interfacial tensions at the 0.2% protein/corn oil interface revealed negative correlations with the effective hydrophobicities determined by both methods indicating lower interfacial tensions with more hydrophobic proteins.     

The relationship between hydrophobicity and interfacial tension of proteins

Charge-free hydrophobic gels of Hjerten et al. (Hjerten, S., Rosengren, J. and Pahlman, S. (1974) J. Chromatogr. 101, 281–288) were used for hydrophobic affinity chromatography. The effective hydrophobicity of proteins was expressed as their retention volumes from columns of butylepoxy- and hexylepoxy-Sepharose 4B. The effective hydrophobicity was also estimated by a partition method of Shanbhag and Axelsson ((1975) Eur. J. Biochem. 60, 17–22) from the partition coefficients of proteins between two phases, poly (ethylene glycol) and dextran. The former contained a hydrophobic ligand, palmitate.A close correlation was observed between the hydrophobicities determined by the two methods. However, no significant relationship was observed between these effective hydrophobicities and the average hydrophobicity of Bigelow ((1967) J. Theoret. Biol. 16, 187–211) that was calculated from the total amino acid composition of each protein.The interfacial tensions at the 0.2% protein/corn oil interface revealed negative correlations with the effective hydrophobicities determined by both methods indicating lower interfacial tensions with more hydrophobic proteins.     

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)–sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH 7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600–salt and PEG-8000–salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein–ion and protein–solvent interactions in the phases of ATPS are primarily important for protein partitioning.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial

Summary In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2–6×10⁶ sec^–1 at 22°C is calculated suggesting a channel mechanism.We would like to thank Kate Van Fossen for her faithful technical support. This work was supported by NIH grants No. HL-20985 and HL-37593.