The effect of pH on heat denaturation and gel forming properties of soy proteins

This study is focussed on the influence of pH on the gel forming properties of soy protein isolate and purified glycinin in relation to denaturation and aggregation. At pH 7.6 more fine-stranded gels were formed characterised by low G' values, and a smooth, slightly turbid appearance, whereas at pH 3.8 coarse gels were obtained with a high stiffness and a granulated, white appearance. Low G' values, as found at pH 7.6, correlate with a high solubility of glycinin and soy protein isolate (ca. 50%) after heating at low protein concentration. At pH 3.8 all protein precipitated upon heating, which correlates with relatively high G' values. The role of beta-conglycinin during gelation of SPI seems to be minor at pH 7.6, which is indicated by the fact that, in contrast to pH 3.8, notable gel formation did not start upon heat denaturation of beta-conglycinin. Furthermore, the mechanism of gel formation seems to be affected by pH, because at pH 7.6, in contrast to pH 3.8, the disulphide bridge between the acidic and the basic polypeptide of glycinin is broken upon heating.     

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.     

A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain

Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D_{20, W} = 3.1 × 10^?7 cm² s^?1 and the value of D_{20, W} = 5.1 × 10^?7 cm² s^?1 calculated for tubulin dimer of M_rel 100,000. The 30 S ring has an observed diffusion coefficient of D_{20, W} = 0.49 × 10^?7 cm² s^?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.     

The fractionation of diphtheria toxoid by means of ammonium sulphate

At high toxoid concentrations as well as at low pH values the curves of the salting out process of diphtheria toxoid with ammonium sulphate have the classical S shape. At lower toxoid concentrations and at a pH value a little below 7 or above it, a part of the curve is horizontal or in some cases even descends. This suggests the presence of two flocculating components, one of which (component A) is precipitated at low (NH₄)₂SO₄ concentrations; this component is probably not immunogenic, or only poorly so. Fractionation and gel diffusion experiments confirmed the presence of these two components. In certain fractions of the toxoids the components may manifest themselves separately by the presence of two flocculation zones.For the purpose of an adequate purification of crude diphtheria toxoid by means of salting out, it appears advisable to keep the toxoid concentration below circa 1500 Lf/ml and the pH at a value higher than 6.5.     

A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultracentrifugal Analysis of Staphylococcal Alpha Toxin

Ultracentrifugal examination of staphylococcal alpha toxin at different stages of purification showed the presence of a major component having a sedimentation coefficient of 2.8S, present to the extent of more than 90% of the sample, and identifiable with active toxin. Several minor components having S(20,w) values of 11.5S, 8.5S, and 2.0S were detected. The 11.5S component presumably is identical with a toxin aggregate studied earlier and designated 12S; the 8.5S component appears to be delta toxin. A sedimentation equilibrium study of more highly purified material gave 32,700 as the best estimate of molecular weight of alpha toxin. Lowering the pH of the partially purified alpha toxin from 10.2 to 5.3 resulted in a small increase in S(20,w) of the 11.5S component and in the disappearance of the 8.5S component, whereas the S(20,w), molecular weight, and hemolytic activity of the toxin remained constant. Exposure of toxin to pH 3.5 irreversibly reduced the S(20,w) to 2.0S, the molecular weight to about 16,000, and caused irreversible inactivation. Raising the pH of acid-inactivated toxin and adding sodium dodecyl sulfate to 1% increased the S(20,w) to near its normal value (2.7S) but did not restore activity.     

Effect of Acidity on the Composition of an Indigenous Soil Population of Rhizobium trifolii Found in Nodules of Trifolium subterraneum L

Acidity affected which members of an indigenous soil population of Rhizobium trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO(3) (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.     

Isolation, characterization and oxygen equilibrium of an extracellular haemoglobin from Eunice aphroditois (Passas).

The extracellular haemoglobin from the polychaeta,Eunice aphroditois, existed as a mixture of a heavy major component (so20, w = 56.96 +/- 0.125) and a light minor component (so20, w = 10.00 +/- 0.13S), the latter probably being a dissociation product of the former. The molecular weight of the purified heavier component, as detetermined by sedimentation equilibrium, was 3.44 x 10(6) +/- 0.04x10(6). The molecule had the electron-microscopic appearance typical of annelid haemoglobins, consisting of a stack of two hexagonal plates, with dimensions 26.32 +/- 0.27 nm across the flats of the hexagon, height of stack 17.86 +/- 0.34 nm. The sugar composition is reported, and the isoelectric point was approx. pH7.8. The haem content was 2.31 +/- 0.01%, corresponding to a minimal mol.wt. of 26700. Detergent/gel electrophoresis revealed the presence of at least four bands with molecular weights in the range 14600-31000. Five N-terminal amino acids were found. In addition to the 10S component, which co-existed with the 57S component at all pH values in the range 4.0-10.6, at low pH values (less than pH.5.0) A 16S and a 1.9 S component were found. The absorption and circular-dichroic spectra are reported, and the alpha-helical content, calculated from the ellipticity at 222 nm, was about 40%. The molecule bound O2 co-operatively with a maximum value of the Hill coefficient, h, of 3.9. Over the pH range 7.0-8.0 there was a positive Bohr effect.     

Wettability and bacteria attachment evaluation of multilayer proteases films for biosensor application

Multilayer films were prepared through a self-assembly technique of proteases. Solutions of pepsin, lysozyme and trypsin at 10⁻⁵ M (pH 6.4, pH 6.4, and pH 7.6, respectively) were used as precursors for film building. The wettability of the film surfaces were estimated by contact angle measurements indicating a higher hydrophobicity to trypsin. This was in agreement to the calculated surface tension components. The patterns of the films were examined using atomic force microscopic images. Surfaces before and after bacteria (Escherichia coli) interactions were also characterized. The results indicate that the hydrophobicity plays a key role in bacterial adhesion and that roughness can be considered as a secondary factor.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

To determine the stage at which H⁺ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m-KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C.Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H⁺ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H⁺ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H⁺ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H⁺ ion is bound before any polymerization takes place.Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m-KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m-KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H ⁺ ions are released.These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H⁺ ions, or, alternatively, two-turn helices accompanied by the binding of H⁺ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H⁺ ions.     

Properties of the high-molecular-weight protein (spectrin) from human-erythrocyte membranes.

The high-molecular-weight protein component from human erythrocytes has been isolated and its solubility properties studied. In physiological solvent conditions the spectrin is not aggregated and is unaffected, both in hydrodynamic properties and conformation, as judged by circular dichroism and intrinsic fluorescence, by the addition of calcium ions. When the pH is decreased an opalescence first sets in, which corresponds to an associated fibrous state of the protein, and when a critical pH is reached precipitation ensues. The precipitation profile is characterised by extreme sharpness, of the kind observed in the phase separation of polyacid-polybase mixtures or of polyampholytes. The addition of calcium ions displaces this precipitation edge towards higher pH. Sodium ions have a similar but smaller effect. The position of the profile is significantly displaced in aged spectrin preparations, or those from frozen erythrocyte ghosts. Fresh preparations of spectrin consist predominantly of a component sedimenting at 9.7 S, with a minor component at 4.4 S (and traces of higher aggregates). The pattern is independent of the ionic strength, or of the presence or absence of calcium ions. The proportion of the small component increases with time, and in spectrin preparations from frozen ghosts it invariably predominates. At low concentrations of guanidine hydrochloride the larger component gives place progressively to the smaller, and vanishes completely when the concentration of the denaturant reaches 1 M. The two components coexist at concentrations below this, and are not in rapid interconversion equilibrium. On recovery of the protein from the guanidine hydrochloride by dialysis, the original pattern of two components is regained. On the other hand the larger component is not found in the material recovered from guanidine hydrochloride solutions of preparations that contain only the small component at the outset. The recovered spectrin is similar to the starting material in its circular dichroism, in its pH-precipitation profiles, and the manner in which the latter is modified by calcium ions. Molecular weight determination by sedimentation equilibrium shows that the 4.4-S species has a molecular weight of some 230 000, which is also the value derived from the extrapolated sedimentation coeffiecient in 6 M guanidine hydrochloride, and thus corresponds to single chains (of which two or more species are resolved in acrylamide gel electrophoresis in the presence of sodium dodecylsulphate); the 9.7-S species, which characterises what is evidently the native state of the extracted spectrin, is found to be a dimer. The frictional coefficients of the monomer and dimer are appreciably different. That of the dimer is compatible with a somewhat asymmetric structure, but by no means to the extent expected for a myosin-like or paramyosin-like molecule.     

Effect of Acidity on the Composition of an Indigenous Soil Population of Rhizobium trifolii Found in Nodules of Trifolium subterraneum L.

Acidity affected which members of an indigenous soil population of Rhizobium trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO₃ (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.     

Molecular Construction of Clostridium botulinum Progenitor Toxin

Molecular dissociation of purified type F progenitor toxin with an S20,W of 10.3 and a molecular weight of 235,000 into two components, toxic and atoxic, was demonstrated by ultracentrifugation, gel filtration, and diethylaminoethyl-Sephadex chromatography at pH 7.5. The ultracentrifugal analysis indicated that type F progenitor toxin dissociates into components of the same molecular size of 5.9S. The toxic component contained a toxicity of 2.5 times 10-8 50% lethal doses per mg of N. Much higher stability of progenitor toxin than that of derivative toxin, particularly at pH below 5, suggests that only progenitor toxin can act as an oral toxin.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.     

Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6). At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7. 6)=25.4+/-6.4 microM at pH 7.6. The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6). Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity. (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30).     

Pepsinogen B: the zymogen of pepsin B

1. Pepsinogen B, the precursor of pepsin B, has been isolated by ion-exchange chromatography and gel filtration from neutral extracts of pig gastric mucosa. The material possesses potential activity against acetyl-l-phenylalanyl-l-di-iodotyrosine and against gelatin but has little, if any, potential activity against haemoglobin. 2. The material appears homogeneous in the ultracentrifuge, but on gel filtration and on electrophoresis in starch gel it is shown to be contaminated with a small amount of material having potential activity against haemoglobin. On electrophoresis in starch gel also the material is shown to contain about equal amounts of two major components, both of which have potential activity against the synthetic substrate. Pepsin B has also been shown to contain two active components by electrophoresis under the same conditions. 3. The zymogen is similar to pepsinogen and pepsinogen C in its molecular weight and general physico-chemical properties, but differs from these zymogens in the nature of its N-terminal residues. It is possible that one of the components contains 1 mole of bound phosphate/mole. 4. The material is activated rapidly at pH2 and more slowly at pH4. At both pH values the kinetics of the activation reaction are complex.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38) by the human UDP-glucuronosyltransferases encoded at the UGT1 locus.

7-Ethyl-10-hydroxycamptothecin (SN-38) is a very promising anticancer drug used for the treatment of metastatic colonrectal cancer. SN-38 is the active metabolite of irinotecan, a semisynthetic anticancer drug derived from 20(S)camptothecin. In this study, we examined the potential for each of the UGT1-encoded isoforms (UGT1A1 and UGT1A3 through UGT1A10) to glucuronidate SN-38. The amount of specific protein for each isoform was determined by Western blot analysis. Although UGT1A1 was previously shown to metabolize this drug, the results of this study show that UGT1A7 glucuronidates this chemical at a 9- to 21-fold higher level at pH 6. 4 and pH 7.6, respectively, than that by UGT1A1. The activity of UGT1A7 is from 8.4- to 19-fold higher at pH 6.4 and 12- to 40-fold higher at pH 7.6 than that by the other 7 UGT1 encoded isoforms. UGT1A7 glucuronidates SN-38 with an apparent Km of 5 microM. Hence, the distribution of this isoform in the gastrointestinal tract has the potential to impact the effectiveness of this chemotherapeutic agent.