Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone.     

Discovery of zoniporide: a potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.     

Dioxygen solubility in aqueous phosphatidylcholine dispersions

The solubility of molecular oxygen, or dioxygen, in low weight percent (1.5%) sonicated dimyristoylphosphatidylcholine (DMPC) aqueous dispersions saturated with air has been measured as a function of temperature between 10 degrees C and 40 degrees C. A modified Winkler technique was used involving a dual cell coulometric titration with voltammetric endpoint detection in a mixed solvent (methanol/water). The results indicate that dioxygen is approximately four times more soluble in the liquid crystalline bilayers (above 24 degrees C) than in the gel state bilayers (below 24 degrees C). The solubility of dioxygen in the bilayer does not appear to be strongly temperature dependent on either side of the 24 degrees C phase transition. The dioxygen solubility in gel state DMPC is approximately equal to that in water at the same temperature. Our result are contrasted with recent measurements made using EPR spin labels.     

O2 solubility in aqueous media determined by a kinetic method

A kinetic method for the determination of O2 solubility in air-saturated aqueous solutions of widely varying composition and temperature is described. It is based on the precise molar stoichiometry between the rates of uptake of H+ and O2, measured with response-matched electrodes, in the reaction NADH + H+ + 1/2O2----NAD+ + H2O, catalyzed by an NADH oxidase preparation. To the initially anaerobic test system, which contains an excess of NADH and NADH oxidase in a buffered medium, an aliquot of the O2-containing solution to be tested is added and the rates of both O2 uptake and H+ uptake are recorded; the H+ electrode is calibrated against standard HCl. From these data the amount of O2 in the aliquot is calculated. Some representative values for O2 solubility at 25 degrees C and 760 mm in air-saturated systems are (i) distilled H2O, 516 nmol O/ml, (ii) 0.15 M KCl, 480 nmol O/ml, and (iii) 0.25 M sucrose, 458 nmol O/ml. Data and equations are also given for the solubility of O2 at 760 mm in air-saturated and lightly buffered 0.15 M KCl and 0.25 M sucrose over the range 5 to 40 degrees C. In the method described the rates of O2 and H+ uptake are precisely linear and stoichiometric when NADH is present in large excess over O2. However, when O2 is in excess and small additions of 340-nm-standardized NADH are made, as in earlier methods based on NADH oxidation, the endpoint is approached very gradually and tends to overestimate O2 solubility, owing to (i) the higher Km for NADH than for O2, (ii) the relatively slow response of the Clark O2 electrode, and (iii) the incomplete oxidation of NADH in the presence of 340-nm-absorbing inhibitory substances.     

Crystal structure and aqueous solubility of ammonium D-glucarate

Ammonium D-glucarate, NH(4)(C(6)H(9)O(8)) [ammonium D-saccharate, NH(4)-SAC], has been synthesized, and its crystal structure solved by single-crystal X-ray diffraction methods. NH(4)-SAC crystallizes in the monoclinic space group P2(1) (#4) with cell parameters a = 4.8350(4) Angstroms, b = 11.0477(8) Angstroms, c = 16.7268(12) Angstroms, beta = 90.973(1) degrees, V = 894.34(12) Angstroms(3), Z = 3. The structure was refined by full-matrix least-squares on F(2) yielding final R-values (all data) R1 = 0.0353 and R(w)2 = 0.0870. The structure consists of alternating (NH(4))(+) and (C(6)H(11)O(6))(-) layers parallel to the bc plane. An extended network of N-H...O(SAC) and O(SAC)-H...O(SAC) hydrogen bonds provide the 3-D connectivity. The aqueous solubility (S(w)) has been shown to be pH independent at ambient conditions within the range 4.5 < pH < 10 with S(w) = 2.19 M/L, whose value is about a factor of two lower than that of the ammonium isosaccharate analogue.     

Physico-chemical aspects of solubility of myosin in aqueous media

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.     

Study of oxytetracycline solubility in an aqueous medium]

Solubility of oxytetracycline dihydrate in aqueous media was studied. It was shown that solubility of the drug in bidistillate at a temperature of 20 degrees was 195 gamma/ml, which was much lower than the requirements of some pharmacopoeia with respect to the drug solubility. Dependence of oxytetracycline dihydrate solubility in the aqueous medium on the values of pH and temperature was found. Indigency of the procedure described in the literature for determination of antibiotic solubility according to the dry weight of the filtrate on addition of large excesses of the solid phase to the system was shown.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.     

Improvement in aqueous solubility achieved via small molecular changes

Overcoming poor solubility is a significant issue in drug discovery. The most common solution is to replace carbon atoms with polar heteroatoms such as N and O or by attaching a solubilizing appendage. This approach can lead to other issues such as poor activity and PK or the increased risk for toxicity. However, there are more subtle structural changes which can be employed that lead to an increase in solubility. These include, excising hydrophobic groups which do not efficiently contribute to binding, modifying stereo- and regiochemistry, increasing or decreasing the degree of unsaturation or adding small hydrophobic groups such as fluorine or methyl.     

3,4-Diaryl-5-hydroxyfuranones: highly selective inhibitors of cyclooxygenase-2 with aqueous solubility

The introduction of a hydroxyl group into the 5-position of the diaryl furanone system provides highly selective inhibitors of cyclooxygenase-2. These molecules can be converted into their sodium salts which are water soluble, facilitating intravenous formulation. These salts show excellent potency in rat models of pain, fever and inflammation.     

Enhancing the aqueous solubility of d4T-based phosphoramidate prodrugs

A range of polyether para-substituted phosphoramidates were synthesised and found to have substantially elevated aqueous solubilities compared to the underivatised parent prodrug. A 30-fold increase in aqueous solubility could be achieved without a substantial decrease of in vitro activity against HIV-1. Replacement of the aryl (i.e. phenolic) moiety by tyrosine led to a substantial enhancement in aqueous solubility but also to a decrease in antiviral potency. A previously unobserved trend was identified, relating increased aryl substituent steric bulk to decreased antiviral activity.     

A simple in vivo assay for increased protein solubility.

Low solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Discovery of a potent tubulin polymerization inhibitor: Synthesis and evaluation of water-soluble prodrugs of benzophenone analog

Prodrugs have proven to be very useful in enhancing aqueous solubility of sparingly water-soluble drugs, thereby increasing in vivo efficacy without a need of special excipients. In vitro and in vivo evaluations of a number of amino acid prodrugs of 1, a previously identified potent tubulin polymerization inhibitor and cytotoxic against various cancer cell lines led to the discovery of 3·HCl (l-valine attached) which is highly efficacious in mouse xenografts bearing human cancer. Pharmacokinetic analysis in rats revealed that compound 1 was released immediately upon administration of 3·HCl intravenously, with rapid clearance of 3·HCl indicating the effective cleavage of prodrug. Compound 3·HCl (CKD-516) has now been progressed to phase 1 clinical trial.     

Enhancing penicillin fermentations by increased oxygen solubility through the addition of n-hexadecane

n-Hexadecane was added to fermentation media to increase the medium oxygen solubilities, thus enhancing oxygen transfer rates in penicillin fermentations. For shake flask fermentations, cells were found to grow faster in the flasks with n-hexadecane than those without. The addition of n-hexadecane to penicillin fermentations was shown to significantly increase cell growth and penicillin production and reduce formation of mycelial pellets. The result was attributed to the enhancement of oxygen transfer in mycelial fermentations due to the higher oxygen solubilities of fermentation media achieved by adding n-hexadecane.     

Specifically increased solubility of enzymes in polyethyleneglycol solutions using polymer-bound triazine dyes

The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), present in an extract of Bakers' yeast, are largely kept in solution by minor amounts of polyethylene glycol-bound triazine dyes (Procion yellow HE-3G and Procion olive MX-3G) even when the solution contains such concentrations of polyethylene glycol (12.5% w/w) which normally precipitate the enzymes. The specific prevention from precipitation can be used for purification of enzyme, preferentially in dealing with crude extracts, which has been demonstrated in this work. A 3.4-fold purification of glucose-6-phosphate dehydrogenase has been achieved with good recovery (93%). Further purification has been possible by combining the recovered (enzyme-containing) supernatant liquid with a solution of dextran which generates an aqueous two-phase system. The lower, dextran-containing phase extracts part of the remaining bulk proteins leaving the target enzyme in the upper phase. The advantages of this method for enzyme purification in large scale are discussed.