Effect of cosolvents on solubility of protein in equilibrium between different states

This paper describes a theoretical work on the solubility of a protein in equilibrium between different forms such as isomerization (A ? B) and dimerization (2A ? B). Under the assumptions that A and B have different solubilities and form their own solid phases (in the form of either precipitation or crystal), it was shownthat only either A or B precipitates at a given condition, another form being in equilibrium in solution with the precipitating form, provided that the solubility ratio of A to B is not identical to their equilibrium composition in the solution phase determined by the equilibrium constant. It follows, then, that the total concentration of the protein in the solution phase can be calculated by summing the solubility of the precipitating form and the concentration in solution of another form in equilibrium with the precipitating form. Assuming various preferential protein-solvent interactions for the two forms in solution as well as solid phases, dependences of the total protein solubility on the additive concentration were examined. It was shown that this dependence may be complex, showing maximum, minimum, or inflection point, depending on the preferential protein interactions with solvent components.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.     

THE CONDITIONS OF RECOVERY OF TRANSMISSIVITY OF NEWLY REPASSIVATED IRON WIRES IN NITRIC ACID

1. Passive steel wires were activated in a bath (Bath A) containing 70 v. per cent HNO₃ (in which they undergo prompt repassivation), and immediately transferred to a second bath (Bath B) containing HNO₃ of a concentration varying in different experiments. After varying intervals in this bath they were transferred while still passive to a third bath (Bath C) containing strong HNO₃ (70 or 100 v. per cent) and there immediately activated. 2. During the immersion in Bath B the wires progressively recover their ability to transmit activation waves in strong HNO₃. The measure of this recovery is the distance travelled by the activation waves in Bath C after the varying times of exposure in Bath B. Transmissivity as thus measured is at first incomplete (decremental) and later becomes complete. The minimal exposures in Bath B required to render wires completely transmissive in the strong acid of Bath C were determined for concentrations of HNO₃ between 10 and 100 v. per cent. With 100 v. per cent HNO₃ in Bath C, these exposures range from 40 minutes or more in 15 v. per cent to 10 minutes in 100 v. per cent HNO₃ (temperature 19–20° in all baths). 3. The time required for complete recovery varies inversely with the concentration of the acid in the recovery bath (Bath B), but increases rapidly with the concentration of the acid in the testing bath (Bath C). Hence at a time when a wire has recovered just sufficiently to transmit non-decrementally in a given strong acid (e.g., 70 v. per cent) it still transmits decrementally in a stronger acid. Complete recovery for transmission in 100 v. per cent HNO₃ requires about twice as long as for 70 v. per cent HNO₃. In HNO₃ of 50 v. per cent and less decremental transmission does not occur. 4. The indications are that recovery is an effect of the progressive solvent action of the external acid on the passivating oxide film, which at its first deposition appears to be relatively thick and hence resistant to electrochemical reduction. The final stage of recovery, when electrical sensitivity and speed of transmission are maximal, would on this hypothesis correspond to minimal thickness, possibly monomolecular. 5. The rate of recovery in Bath B is not far from proportional to the concentration of HNO₃ in the more dilute solutions, but in the higher, especially the strongly passivating, concentrations (70 to 100 v. per cent) the rate becomes appreciably slower than proportional, apparently because of the intense oxidizing action of these solutions, which reinforces the oxide sheet and retards the thinning process. 6. The bearing of these observations on the problem of the conditions of recovery in irritable living tissues (such as nerve) during the absolute and relative refractory periods is briefly discussed.     

MOLECULAR ASSOCIATION OF HEMOCYANIN PRODUCED BY X-RAYS AS OBSERVED IN THE ULTRACENTRIFUGE

1. When normal, monodisperse hemocyanin (60.5S) from Limulus Rolyphemus was irradiated in neutral buffer with x-rays, several new, more rapidly sedimenting ultracentrifugal components (86S, 107S, 122S) were produced, with a corresponding loss in the amount of the unaffected protein. The amount of the effect was roughly proportional to the amount of irradiation. 2. The new resolvable components apparently represented an association of the primary particles into aggregates of 2, 3, and 4 primary particles respectively. 3. The proportional amount of hemocyanin affected decreased almost to the vanishing point as the concentration of the protein was raised to high levels. 4. The absolute effect, i.e. the total number of particles affected in a given volume, increased with the concentration of hemocyanin, at least for concentrations below 15 per cent. 5. The presence of 33 per cent horse serum during irradiation inhibited the effect on the hemocyanin almost completely, with hemocyanin concentrations of both 0.8 and 14 per cent. 6. The presence of 2.8 per cent egg albumin during irradiation lowered the effect by about 70 per cent in the case of dilute preparations (0.8 per cent hemocyanin), but by only about 25 per cent in the case of 14 per cent solutions. 7. A lowering of the solution''s oxygen tension during irradiation enhanced the effect, almost doubling it in some cases. 8. The probable theoretical significance of these and other observations are discussed in the text.     

Protein crystallography at sub-zero temperatures: lysozyme-substrate complexes in cooled mixed solvents.

To determine the feasibility of direct X-ray crystallographic structure determination of productive enzyme-substrate complexes and to ascertain the best conditions for such studies, the hydrolysis of bacterial cell walls and oligosaccharides by human leukaemic lysozyme was investigated in mixed aqueous/organic solvents and high salt solutions. Although high salt solutions modify the enzymic reaction, hydrolysis in mixed solvents appears to proceed by the same mechanism as in aqueous solution. At low temperatures the reaction is slowed progressively, and at −25 °C the enzyme-substrate complex in mixed solvents is stable indefinitely. The conformation of the enzyme is not significantly altered in these solvents, and the enzyme-substrate complex can be formed by direct addition of substrate to the enzyme at sub-zero temperatures, as required for crystallographic studies. The pH profile of the reaction in mixed solvents allows conditions of optimal binding to be selected. These studies in solution demonstrate that low-temperature protein crystallography may indeed permit the direct determination of the three-dimensional structure of enzyme-substrate complexes. They also delineate the precise conditions of pH, temperature and solvent to use in the crystallographic experiments.     

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity.     

A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa

A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

CRYSTALLINE ACETYL DERIVATIVES OF PEPSIN

Crystalline pepsin has been acetylated by the action of ketene in aqueous solution at pH 4.07–5.5. As acetylation proceeds the activity decreases, the decrease being more rapid at pH 5.0–5.5 than at 4.0–4.5. Three acetyl derivatives have been isolated from the reaction mixture and obtained in crystalline form. The crystal form of these derivatives is similar to that of pepsin. Fractionation and solubility determinations show that these preparations are not mixtures or solid solutions of the original pepsin with an inactive derivative. A compound which contains three or four acetyl groups and which has lost all of its original primary amino groups can be isolated after short acetylation. It has the same activity as the original pepsin. A second derivative containing six to eleven acetyl groups has also been isolated. It has about 60 per cent of the activity of the original pepsin. A third derivative having twenty to thirty acetyl groups and about 10 per cent of the activity of original pepsin can be isolated after prolonged acetylation. The 60 per cent active derivative on standing in strong acid solution loses some of its acetyl groups and at the same time regains the activity of the original pepsin. The compound obtained in this way is probably the same as the completely active three acetyl derivative obtained by mild acetylation. These results show that acetylation of three or four of the primary amino groups of pepsin causes no change in the specific activity of the enzyme but that the introduction of acetyl groups in other parts of the molecule results in a marked loss in activity. The solubilities, amino nitrogen content, acetyl content, isoelectric point, and the specific activity have been determined by a variety of methods and found to be different from the corresponding properties of crystalline pepsin. The pH-activity curves, acid and alkali inactivation, and titration curves were not significantly different from the same respective properties of pepsin.     

The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides: II. Studies on Poly-l-Glutamic Acid

The Moffitt b₀ parameter of poly-L-glutamic acid in the presumed helical state varied with solvent composition, ranging in magnitude from less than 600° in aqueous solution to 800° in methanol. b₀ was also dependent on temperature throughout the excessable temperature range. The value in aqueous solution is at least 100° smaller than the values for a number of polypeptides in organic solvents, when compared at the same refractive index. Therefore the optical rotatory dispersion data do not provide evidence that the molecule is completely helical in aqueous solution. Since other types of evidence for helical content are not sufficient to establish that PLGA is a complete helix, the helical content of proteins and polypeptides determined by rotatory dispersion measurements should be regarded as uncertain by about 20 per cent.     

THE RELATION BETWEEN THE ELECTRICAL CONDUCTIVITY OF THE EXTERNAL MEDIUM AND THE RATE OF CELL DIVISION IN SEA URCHIN EGGS

Dilution of sea water with isotonic sugar solution leaves the rate of cleavage of Arbacia eggs almost unchanged until the proportion of sea water is decreased to 20 or 25 volumes per cent. From this point cleavage becomes progressively slower with further dilution. Many eggs fail to cleave at dilutions of 5 to 6 volumes per cent. No cleavage occurs in 2 volumes per cent sea water or in pure sugar solution. Eggs returned from these media to sea water resume cleavage and development. There is thus no relation between the rate of cleavage and the electrical conductivity of the medium, except possibly within the range of dilutions from 20 to 5 volumes per cent sea water. In this range cleavage rate decreases as conductivity decreases, but the relation is not a linear one.     

THE INFLUENCE OF ELECTROLYTES ON THE SOLUTION AND PRECIPITATION OF CASEIN AND GELATIN

1. Colloids have been divided into two groups according to the ease with which their solutions or suspensions are precipitated by electrolytes. One group (hydrophilic colloids), e.g., solutions of gelatin or crystalline egg albumin in water, requires high concentrations of electrolytes for this purpose, while the other group (hydrophobic colloids) requires low concentrations. In the latter group the precipitating ion of the salt has the opposite sign of charge as the colloidal particle (Hardy''s rule), while no such relation exists in the precipitation of colloids of the first group. 2. The influence of electrolytes on the solubility of solid Na caseinate, which belongs to the first group (hydrophilic colloids), and of solid casein chloride which belongs to the second group (hydrophobic colloids), was investigated and it was found that the forces determining the solution are entirely different in the two cases. The forces which cause the hydrophobic casein chloride to go into solution are forces regulated by the Donnan equilibrium; namely, the swelling of particles. As soon as the swelling of a solid particle of casein chloride exceeds a certain limit it is dissolved. The forces which cause the hydrophilic Na caseinate to go into solution are of a different character and may be those of residual valency. Swelling plays no rôle in this case, and the solubility of Na caseinate is not regulated by the Donnan equilibrium. 3. The stability of solutions of casein chloride (requiring low concentrations of electrolytes for precipitation) is due, first, to the osmotic pressure generated through the Donnan equilibrium between the casein ions tending to form an aggregate, whereby the protein ions of the nascent micellum are forced apart again; and second, to the potential difference between the surface of a micellum and the surrounding solution (also regulated by the Donnan equilibrium) which prevents the further coalescence of micella already formed. This latter consequence of the Donnan effect had already been suggested by J. A. Wilson. 4. The precipitation of this group of hydrophobic colloids by salts is due to the diminution or annihilation of the osmotic pressure and the P.D. just discussed. Since low concentrations of electrolytes suffice for the depression of the swelling and P.D. of the micella, it is clear why low concentrations of electrolytes suffice for the precipitation of hydrophobic colloids, such as casein chloride. 5. This also explains why only that ion of the precipitating salt is active in the precipitation of hydrophobic colloids which has the opposite sign of charge as the colloidal ion, since this is always the case in the Donnan effect. Hardy''s rule is, therefore, at least in the precipitation of casein chloride, only a consequence of the Donnan effect. 6. For the salting out of hydrophilic colloids, like gelatin, from watery solution, sulfates are more efficient than chlorides regardless of the pH of the gelatin solution. Solution experiments lead to the result that while CaCl₂ or NaCl increase the solubility of isoelectric gelatin in water, and the more, the higher the concentration of the salt, Na₂SO₄ increases the solubility of isoelectric gelatin in low concentrations, but when the concentration of Na₂SO₄ exceeds M/32 it diminishes the solubility of isoelectric gelatin the more, the higher the concentration. The reason for this difference in the action of the two salts is not yet clear. 7. There is neither any necessity nor any room for the assumption that the precipitation of proteins is due to the adsorption of the ions of the precipitating salt by the colloid.     

PURIFICATION AND CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN

Purified preparations of diphtheria antitoxin have been obtained by digestion of the toxin-antitoxin complex with trypsin, followed by fractional precipitation with ammonium sulfate. The various fractions obtained in this way are all 90 per cent or more precipitated by diphtheria toxin but combine with different quantities of the toxin. The fraction precipitated between 0.33 and 0.5 saturated ammonium sulfate is homogeneous by electrophoresis and ultracentrifuge but does not have constant solubility. A small amount of a more soluble fraction has been obtained which does have constant solubility and satisfies the criteria of a pure protein. This protein crystallizes readily in poorly formed thin plates. It is very unstable and reverts to a less soluble non-crystallizable form. It has a sedimentation constant of 5.7 x 10^–13 and a molecular weight of 90,500. It has an antitoxic value of 700–900 flocculation units per mg. protein nitrogen and has an antitoxic value by the protection test of about 700 units per mg. protein nitrogen. The precipitation range of the purified antitoxin with purified toxin is much wider than that with crude preparations.     

Protein purification with vapor-phase carbon dioxide

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.     

Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.     

Site‐to‐site variability and temporal trends of DOC concentrations and fluxes in temperate forest soils

Here, we report site‐to‐site variability and 12–14 year trends of dissolved organic carbon (DOC) from organic layers and mineral soils of 22 forests in Bavaria, Germany. DOC concentrations in the organic layer were negatively correlated with mean annual precipitation and elevation whereas air temperature had a positive effect on DOC concentrations. DOC fluxes in subsoils increased by 3 kg ha^?1 yr^?1 per 100 mm precipitation or per 100 m elevation. The highest DOC concentrations were found under pine stands with mor humus. Average DOC concentrations in organic layer leachates followed the order: pine>oak>spruce>beech. However, the order was different for mean DOC fluxes (spruce>pine>oak>beech) because of varying precipitation regimes among the forest types. In 12 of 22 sites, DOC concentrations of organic layer leachates significantly increased by 0.5 to 3.1 mg C L^?1 yr^?1 during the sampling period. The increase in DOC concentration coincided with decreasing sulfate concentration, indicating that sulfate concentration is an important driver of DOC solubility in the organic layer of these forest sites. In contrast to the organic layer, DOC concentrations below 60 cm mineral soil depth decreased by <0.1–0.4 mg C L^?1 yr^?1 at eight sites. The negative DOC trends were attributed to (i) increasing adsorption of DOC by mineral surfaces resulting from desorption of sulfate and (ii) increasing decay of DOC resulting from decreasing stabilization of DOC by organo‐Al complexes. Trends of DOC fluxes from organic layers were consistent with those of DOC concentrations although trends were only significant at seven sites. DOC fluxes in the subsoil were with few exceptions small and trends were generally not significant. Our results suggest that enhanced mobilization of DOC in forest floors contributed to the increase of DOC in surface waters while mineral horizons did not contribute to increasing DOC export of forest soils.     

The Effect of Subjecting Peas to Air Enriched with Carbon Dioxide: II. RESPIRATION AND THE METABOLISM OF THE MAJOR ACIDS

Whole peas at about 75 per cent of their maximum fresh weightwere subjected to 5–30 per cent CO₂ in air for periodsof from 1–6 d, then returned to air for a further 1 d.Samples were withdrawn at intervals and organic acids, ^TCO₂and ethanol estimated as well as the rate of respiration. Slicesof cotyledons suspended in water were also subjected to highconcentrations of CO₂ in air for 3 h. The rate of respiration was inhibited progressively by increasein CO₂ content of the tissue. The high internal CO₂ contentof the intact pea causes an inhibition of its rate of respirationby about 25 per cent. Alcohol production commenced at between10 and 15 per cent CO₂ in the ambient gas and slowly increasedin rate up to 37 per cent. The CO₂-air mixtures reduced the content of malate, pyruvateand -oxoglutarate, increased that of succinate and left citrateunaffected. On return to air malate rose rapidly and succinatefell slowly to their original concentrations. During the sameperiod the concentration of PEP fell sharply and after about1 h rose again, whereas oxalacetate showed a reverse response.It is argued that the rapid re-synthesis of malate was by carboxylationof PEP to oxalacetate and that this reaction was stimulatedby a change in pH rather than by the direct effect of the changein concentration of CO₂. In one experiment ¹⁴CO₂ was supplied for 2 h before return toair and the movement of ¹⁴C followed for 6 h. The results supportthe method of re-synthesis of malate proposed.     

Studies on immobilized trypsin in high concentrations of organic solvents.

Trypsin was covalently immobilized on porous glass in the presence and absence of a specific substrate and reacted in various organic solvents of different dielectric constants. Optimum solvent concentration, pH profile, Km(app), Vmax(app), productivity versus temperature, activity, and reaction rates were determined. Reaction rates of six lysyl dipeptides were compared. Crystalline trypsin was dansylated for studies by nanosecond fluorescence techniques to determine the effects of introducing high concentrations of organic solvents on the molecule. The results indicated that greater reaction rates were observed with dipeptides having more acidic carboxyl terminal groups. The data also indicated that greater reaction rates were observed in higher concentrations of solvents of lower dielectric constants. Nanosecond fluorescence spectroscopy of trypsin in high concentrations of a low dielectric constant solvent indicated major dehydration even though maximal enzyme activity was achieved under these conditions.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

METABOLISM OF POLYAMINES IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN AND SPLEEN

Abstract— Following intracerebral inoculation of mouse adapted scrapie agent into mice, polyamine concentration in the brain decreases to about 75 per cent of the normal level during the first 2 months after intracerebral inoculation of the agent. Between 2 and 4 months after infection thelevel of spermidine and spermineincreased by 80 and 40 percent respectively to reach concentrations of 25 and 20 per cent higher than controls of the same age. During the same period the rate of incorporation of [¹⁴C]putrescine into spermidine is increased four-fold as compared with controls. The changes in polyamine levels correlate well with the pattern of astrocyte hypertrophy and are similar to those reported for human brain tumours. The concentration of polyamines in spleen increases soon after inoculation. Whilst changes in brain polyamines might be referred to the hypertrophic growth of astrocytes those in spleen are perhaps due to an increased metabolic activity of spleen cells associated with the replication of the agent. These results are derived from experimental mouse scrapie and not naturally occurring disease in sheep.