Granulocyte-colony stimulating factor maintains a thermally stable,compact, partially folded structure at pH 2

At acidic pH many proteins exist in a partially unfolded form, called the “A” state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.O. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stake's radius even at pH 2.0.The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some α-helical structure persists even at 86°C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the “A” state.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrsoine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the trytophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn²⁺-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultraviolet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77° was determined upon heating the enzyme at pH 7.5 in the presence of Mn²⁺-ions for 10 min; in the presence of EDTA, Tm is shifted to 55°. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn²⁺-ion binding.     

Non-native Intermediate Conformational States of Human Growth Hormone in the Presence of Organic Solvents

Purpose. Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.Methods. The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.Results. CD data suggested that the secondary structure of hGH remained unchanged up to 50\% (v/v) ethanol, but the tertiary structure was perturbed at ã20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan.Conclusions. The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30–60% ethanol.     

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.     

Preformulation Study of Highly Purified Inactivated Polio Vaccine,Serotype 3

To improve the effectiveness of the polio vaccination campaign, improvements in the Thermal Stability of IPV3 at pH 7 as the vaccine are being investigated. Here, inactivated polio vaccine, serotype 3 (IPV3) was characterized via a number of biophysical techniques. The size was characterized by transmission electronic microscopy and light scattering. The capsid protein conformation was evaluated by intrinsic fluorescence and circular dichroism (CD), and the D-antigen content by enzyme-linked immunosorbent assay (ELISA). The pH thermal stability of IPV3 (pH 3.0–8.0; 10°C–87.5°C) was evaluated by fluorescence, CD, and static light scattering. The transition temperatures reflect the responses, respectively, of tertiary structure, secondary structure, and size to applied thermal stress. The data were summarized as empirical phase diagrams, and the most stable conditions were found to be pH 7.0 with temperature lower than 40°C. CD detected a higher transition temperature for capsid protein than that for RNA. The effects of certain excipients on IPV3 thermal stability and antigen content were evaluated. The results of their effects, based on intrinsic fluorescence and ELISA, were in good agreement, suggesting the feasibility of applying intrinsic fluorescence as a high-throughput tool for formulation development. The study improves the understanding of IPV3 thermal stability, and provides a starting point for future formulation development of IPV3 and other serotypes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:140–151, 2014     

Circular dichroism of reduced and oxidized recombinant human epidermal growth factor

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mm sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of α-helix, β-sheet, and unordered structure. The addition of the α-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of α-helix, at the apparent expense of β-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.     

Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material.

The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60 degrees C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.     

Interaction of phospholipase A2 from Vipera russelli venom with aristolochic acid: a circular dichroism study

The interaction of aristolochic acid, an alkaloid from Aristolochia species, with phospholipase A2 (PLA2) from Vipera russelli venom was followed by circular dichroism measurements. Aristolochic acid is a non-competitive inhibitor of PLA2. The binding of aristolochic acid to PLA2 induces an extrinsic CD band at 320 nm. The association constant was determined by following the intensity of the extrinsic CD band. Aristolochic acid forms a 1:1 complex with PLA2, with an association constant K, of 5.4 X 10(3) M-1 and a Gibb's free energy change (delta G0) for the reaction of -5.1 kcal/mole. The values of association constant and delta G0 suggest that the interaction is weak. Binding of aristolochic acid causes a change in the secondary structure of the protein which is characterized by an increase in the apparent content of alpha-helix, without any detectable change in the tertiary structure of PLA2.     

Conformational mapping of the N‐terminal segment of surfactant protein B in lipid using 13C‐enhanced Fourier transform infrared spectroscopy

Abstract: Synthetic peptides based on the N‐terminal domain of human surfactant protein B (SP‐B_1?25; 25 amino acid residues; NH₂‐FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full‐length, 79‐residue protein. Here, we used physical techniques to examine the secondary conformation of SP‐B_1?25 in aqueous, lipid and structure‐promoting environments. Circular dichroism and conventional, ¹²C‐Fourier transform infrared (FTIR) spectroscopy each indicated a predominateα‐helical conformation for SP‐B_1?25 in phosphate‐buffered saline, liposomes of 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol and the structure‐promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant β‐ and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope‐enhanced FTIR spectroscopy was performed with 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol liposomes and a suite of SP‐B_1?25 peptides labeled with ¹³C‐carbonyl groups at either single or multiple sites. Combining these ¹³C‐enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP‐B_1?25 in 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol: β‐sheet (residues 1–6), α‐helix (residues 8–22) and random (residues 23–25) conformations. Analogous structural motifs are observed in the corresponding homologous N‐terminal regions of several proteins that also share the ‘saposin‐like’ (i.e. 5‐helix bundle) folding pattern of full‐length, human SP‐B. In future studies, ¹³C‐enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane
environments.     

Conformational effects in the interaction of phenylbutazone with albumin studied by circular dichroism.

The binding of phenylbutazone (PB) to human serum albumin (HSA) at different pH and in the presence of different NaSCN and urea concentrations that alter the conformation of the protein was examined qualitatively on the basis of extrinsic elliptical strength at 288 nm by means of circular dichroism (CD). The values of the binding index expressed as a ratio of [theta]max/[theta]pH7.4(288) at each extrinsic rotational strength in the presence of various concentrations of NaSCN, urea and hydrogen ion were directly proportional to the alpha-helix content based on the peptide backbone alteration of HSA by NaSCN, urea and hydrogen ion except for the pH range of 5.0 to 10.0. The values in the pH range of 7.4 to 10.0 depended on the concentration of hydrogen ion and not on the alpha-helix content, showing a significant effect of the hydrogen ion on the tertiary conformation with respect to the binding sites of the amino acid chain rather than the peptide backbone of HSA. The increases in the binding index observed in the pH range of 7.4 to 10.0 were not observed at all in the case of NaSCN and urea at the concentrations studied. It was demonstrated that the binding of PB to HSA increased with the change in the tertiary conformation caused by hydrogen ions but decreased with that in the secondary conformation caused by a concentration change of NaSCN and urea. Thus, the binding was closely associated with skeletal conformational alterations as well as changes in the binding sites of the amino acid chains of the protein.     

四环类抗生素的反相制备层析——Ⅰ.应用硅胶RP-2层析分离四环类抗生素

反相硅胶是一种新的层析介质,近年来已广泛应用于天然产物的分离和纯化中,为确证反相硅胶RP-2能否有效地分离四环类抗生素,研究了不同溶剂、pH、酸类条件下四环素、金霉素、甲烯土霉素和强力霉素在硅酸RP-2柱上的洗脱特性。据此确定了硅胶RP-2柱层析分离四环素、金霉素,甲烯土霉素和强力霉素的条件。虽然甲烯土霉素和强力霉素在此条件下 难以分离,但应用醋酸乙酯饱和的水(pH6)或醋酸乙酯饱和的草酸溶液(pH3)作洗脱溶剂能有效地从上述四种四环类抗生素中分离出四环素和金霉素。随着草酸浓度的增加和pH的降低,甲烯土霉素和强力霉素也能得到较好的分离。醋酸乙酯饱和的0.05mol/L草酸溶液(pH3)则是从上述四种抗生素中分离出四环素和金霉素的最适宜的溶剂系统。这是一快速、简便的制备性分离四环类抗生素的方法。并且使用过的硅胶RP-2经过适当处理之后还可反复使用。     

Elucidation of two major aggregation pathways in an IgG2 antibody

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 37 degrees C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH - 6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species (approximately 26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH - 5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH - 4.0 and 6.0. The dimer species formed at pH - 5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH - 6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH - 4.0 have altered secondary and tertiary structure.     

Temperature- and pH-Induced Multiple Partially Unfolded States of Recombinant Human Interferon-α2a: Possible Implications in Protein Stability

PURPOSE: To study the effect of solution conditions on the structural conformation of recombinant human interferon-alpha2a (IFNalpha2a) to investigate its tendency to form partially unfolded intermediates. METHODS: The structural properties of IFNalpha2a were studied at various pH values (2.0-7.4) and temperatures (5 degrees C-80 degrees C) using Trp fluorescence emission, fluorescence quenching, near- and far-UV circular dichroism (CD) spectroscopy, and DSC. RESULTS: Fluorescence intensity measurements as a function of temperature indicated the onset of the thermal unfolding of IFNalpha2a, denoted by Td, around 60 degrees C above pH 4.0. Td was not observed at pH 3.5 and below. Acrylamide and iodide quenching studies indicated partial unfolding of protein with decrease in pH and with increase in temperature up to 50 degrees C. Near-UV CD studies indicated a significant loss in the tertiary structure of protein on increase in temperature from 15 degrees C to 50 degrees C at all solution pHs. DSC scans supported results obtained from fluorescence and CD studies at pH 4.0 and below. DSC, however, was insensitive to changes that occurred at moderate temperatures at pH 5.0 and 7.4. CONCLUSIONS: IFNalpha2a has a tendency to acquire multiple partially unfolded states with structural conformations sensitive to solution pH and temperature. These states were formed at moderate temperatures, and it is speculated that these partially unfolded states could play an important role in the aggregation of proteins during the long-term storage of aqueous protein formulations.     

Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony stimulating factor

Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37 degrees C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37 degrees C than at 25 degrees C and partially counteracted by 1.0 M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37 degrees C than at 25 degrees C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H-D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H-D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH.     

Evaluation of a Raman Chemometric Method for Detecting Protein Structural Conformational Changes in Solution

Raman scattering shows promise as a powerful routine tool, to determine both secondary and the smaller tertiary structural changes that precede aggregation in both solutions and solids. A method was developed utilizing principal component analysis (PCA) of Raman spectra for detection of small, but meaningful, pH induced changes in tertiary protein structure linked to aggregate formation using α-lactalbumin solutions as a model. The sample preparation and spectral parameters, were optimized for a bulk Raman probe. Analysis of large regions (600–1850 cm^?1) yielded principal component (PC) scores useful for semi-quantitative comparison of protein conformation between formulations. PC loadings corresponded to specific structural peaks known to change with solution pH. PCA of circular dichroism (CD) spectra of dilute solutions yielded similar results. Sucrose is a common formulation excipient with a Raman spectrum that overlaps many protein peaks. With sucrose in the protein solution, the ability of PCA to discern protein structural changes from the Raman spectra was somewhat reduced. Analysis of a more limited spectral region (1530–1780 cm^?1) with negligible sucrose spectral contribution improved the discrimination of protein conformational states. The new Raman method accurately distinguished differences in protein structure in concentrated solutions. The long-term goal is to explore Raman characterization as a routine monitoring tool of protein stability in both solution and solid states.     

Are secondary structures secondary?

Crystallographically observed secondary structures in globular proteins are divided into two categories based on calculated potential values for these structures. Type I secondary structures are those which have maximum potential in observed structural state. These type I structures consist of those amino acids which intrinsically prefer that structural state. On the other hand type II secondary structures have a calculated potential value which is not maximum for the observed state. Type II structures are composed of those amino acid residues which do not intrinsically prefer the observed state and thus seem to have been formed due to tertiary interactions. All the observed secondary structures have been identified as type I or type II in 31 different globular proteins considered. It is suggested that type II structures are formed at a latter stage of protein folding mainly due to favourable tertiary interactions.     

Modification of bovine serum albumin structure following reaction with 4,5(E)-epoxy-2(E)-heptenal

Bovine serum albumin (BSA) was incubated for different periods of time and in the presence of several concentrations of 4, 5(E)-epoxy-2(E)-heptenal, at pH 7.4 and 37 degrees C, in an effort to analyze the changes produced in its structure as a consequence of its reaction with this product of lipid oxidation. The epoxyalkenal modified the primary structure of BSA as determined by lysine losses and formation of oxidative stress product epsilon-N-pyrrolylnorleucine (Pnl), which depended on the concentration of the aldehyde and the incubation time. These changes also modified secondary and tertiary structures of the protein, which were determined by studying protein denaturation and polymerization. In addition, all these modifications were parallel to the development of color and fluorescence, which were produced as a consequence of the formation and polymerization of pyrrole amino acid residues. The above results indicated that epoxyalkenals modify the protein structure and develop color and fluorescence. A failure in the degradation of these modified proteins might induce their accumulation and, thus, participation in lipofuscin or age pigments formation.     

Secondary structure of a dynamic type I’β‐hairpin peptide

Abstract: A spontaneously folding β‐hairpin peptide (Lys‐Lys‐Tyr‐Thr‐Val‐Ser‐Ile‐Asn‐Gly‐Lys‐Lys‐Ile‐Thr‐Val‐Ser‐Ile) and related cyclic (cyclo‐Gly‐Lys‐Tyr‐Ile‐Asn‐Gly‐Lys‐Ile‐Ile‐Asn) and linear (Ser‐Ile‐Asn‐Gly‐Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D ¹H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The β‐hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 ? i + 2 and i + 2 ?i + 3 interaction, confirming the expected structure. Using NMR α‐proton (H) chemical shifts, the extents of folding of the β‐hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 °C), which was taken to be maximally folded. Substitution of iso‐aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained β‐turns may have adverse effects on secondary structure. N‐terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.     

Effect of pH and Excipients on Structure, Dynamics, and Long-Term Stability of a Model IgG1 Monoclonal Antibody upon Freeze-Drying

Purpose

To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.

Methods

IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR ¹H relaxation was used to monitor glassy state dynamics.

Results

IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.

Conclusions

Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.     

叔胺药物的分配率研究

本文在前二报^(1,2)的基础上进一步研究,讨论了溶剂抽提中有关的四个方面。溶剂:研究了氯仿的混合溶剂。抽提剂:研究了硫酸取代化合物、无机盐及有机盐。被抽提物质:研究了叔胺结构与分配率的关系及pKa与分配率的关系。水相:讨论了在酸碱性各个阶段时对分配率的影响。还讨论了溶剂抽提各方面之间的关系。