Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.     

Chromatographic and enzymatic strategies to reveal differences between amidated pectins on a molecular level

The intermolecular distributions of amide groups within two commercial LMA pectins was studied after removal of the methyl esters followed by fractionation of the different populations by anion exchange chromatography. The populations obtained had almost equal degrees of amidation while the values of the degree of blockiness were not the same, indicating also a different intramolecular distribution of the substituents considered as semirandom. Populations from the methyl-esterified amidated pectins showed a rather random distribution for almost all populations. A striking difference between these different populations was that, despite the same level of substitution, the ratio between amide groups and methyl esters varied significantly, indicating a heterogeneous amidation process.     

Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.     

Analysis of mixtures of pectins and amidated pectins

Amidated pectins and non-amidated pectins are transformed by saponification into amidated pectic acid and pectic acid. By heat treatment under alkaline conditions only amidated pectic acid is depolymerized and can be separated from the high-molecular pectic acid by gel-filtration. On this basis the composition of mixtures can be analysed.     

Structure of citrus pectins and viscometric study of their solution properties

Citrus pectins with degrees of methylation between 30 and 72% were carefully characterized in order to determine their charge density and molecular weight distribution, the content in galacturonic acid and in neutral sugars, the degree of methylation and acetylation. Using enzymic degradation it has been found that pectin molecules consist mainly of long homogalacturonan regions with some regions of neutral sugars as side chains attached on rhamnose residues. The viscometric behaviour of the different samples indicates that 0.1 M NaCl, at 25 degrees C, is a good solvent of sodium pectinates. From the evolution of the Huggins parameter, it appears that pectins with 50% of methylated galacturonic groups exhibit a maximum flexibility. A Mark-Houwink exponent of 0.8 has been found in good agreement with theoretical predictions for flexible polymers in a good solvent.     

Dependence of the catalytic activity of papain on the ionization of two acidic groups.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.     

Intramolecular distribution of carboxyl groups in low methoxyl pectins — A review

Low methoxyl pectins (LMP) have been manufactured since the 1940s primarily for use as gelling agents. At the time, it was noted that low methoxyl pectates (LMPs) prepared by different methods had different gelling properties and this was attributed to the way in which the free carboxyl groups were distributed along the chain following deesterification. Various workers have shown that LMP prepared by enzymic deesterification is more heterogeneous with respect to the degree of esterification (DE) than LMPs of the same DE prepared by acid deesterification. This, together with enzyme studies, has been taken as a sign that pectinesterase works in a sequential fashion and, similarly, acid deesterification is a random process.More recently, the preparation of crude enzyme-deesterified LMP, which is used as a thickener in canned goods, has been described. Although enzyme-deesterified LMPs appear to form weak gels with calcium ions at room temperature (acid-deesterified LMP/calcium gels are reported to be stronger) they are superior when incorporated into canned goods which receive a severe heat treatment.This review briefly describes the preparation of LMP and that work on LMP which provides information about the distribution of carboxyl groups in the pectate molecule. Since it is established that this distribution affects the gelling properties of the pectin a sound understanding of the chemical aspects may assist in understanding the mechanism of gelation.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7–36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7–36)amide and GLP-1(1–36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7–36)amide, GLP-1(1–36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7–36)amide could be identified in the hypothalamus, in amounts that represented 55–94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1–36)amide, representing 0.8–3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7–36)amide (0–0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7–36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27–73% of total GLI, and in five of six specimens only GLP-1(7–36)amide could be identified on HPLC, in amounts representing 13–17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1–36)amide (13% of total GLI) in addition to GLP-1(7–36)amide (31% of total GLI). Acid–ethanol extraction of peptide-free rat plasma with added GLP-1(7–36)amide gave recoveries of 91±SEM 2% in the range 20–200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1±1.1 pmol/l and rose to a maximum of 15.4±3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.     

Hydrolysis of pectins with different degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme

The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme.     

Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin

Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.     

An immunohistochemical localization of neuropeptide Y (NPY) in its amidated form in human frontal cortex

The distribution of neuropeptide Y (NPY)-immunoreactive neurons was studied in human frontal cerebral cortex from surgical biopsy specimens by immunohistochemical techniques. NPY-containing neurons were identified in all cortical sublayers except sublayer I. The stained neurons were of the multipolar, bitufted, round or triangular form with dendritic and axonal processes. The immunoreactive neurons were considered to be cortical interneurons, due to their nonpyramidal form, and since their processes could be followed intracortically particularly in direction to superficial cortical layers. The NPY precursor molecule is processed to NPY by a dibasic cleavage, and NPY is further enzymatically amidated before release and receptor activation can be achieved. Antisera raised against Cys-NPY(32-36)amide recognize amidated NPY not cross-reacting with nonamidated NPY. These antisera and immunohistochemistry revealed the presence of a population of NPYamide-immunoreactive cells morphologically indistinguishable from the NPY-immunoreactive cells in the human frontal cortex. By comparing the number of immunoreactive cells in adjacent sections, it appears that the number of NPY-immunoreactive cells was higher than those immunoreactive to NPYamide. Also, the density of NPY fibers was much higher compared with the number stained with NPYamide antiserum. The present immunohistochemical study indicates that NPY in its amidated form is contained in a subpopulation of human cortical NPY-immunoreactive neurons and may participate as an active neurotransmitter/modulator within the human cerebral cortex.     

Conformations and interactions of pectins: II. Models for junction zones in pectinic acid and calcium pectate gels

Pectinic acid and calcium pectate gels condensed into uniaxially oriented fibers have been studied by X-ray diffraction. Although the diffraction patterns correspond to systems of only limited order, they show that both systems conserve the 1.3 nm axial period and 0.43 nm pseudo-period observed in sodium pectate. Pectinic acid further resembles sodium pectate in packing isometrically in a hexagonal net of side 0.84 nm. On the other hand, calcium pectate fibers contain the 1.2 nm lateral spacing observed in pectic acid. Speculative models for pectinic acid and calcium pectate have been developed. The former structure could be stabilized by hydrophobic binding from columns of methyl groups as well as by specific intermolecular hydrogen bonds. In the latter, the main interactions between pairs of chains could be bridges formed by calcium ions, which incorporate into their co-ordination shells two polyanion oxygen atoms from one chain and three from another. These model-building studies provide plausible visualizations of two different kinds of junction zones that may exist in pectic gels.     

Isolation of 2-Pyruvoylaminobenzamide as an Antiauxin from Colletotrichum lagenarium

To learn of possible contributions of amide side-groups of asparagine and glutamine residues to viscoelastic properties of food proteins, lime-processed gelatin, which has lost these amide side-groups, was amidated by reaction with ammonia and the water soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC). Depending on the quantity of EDC used, 13 to 40.3% of carboxyl side-groups of gelatin was amidated without intra- and inter-molecular cross-linkages, as judged from analyses of free-amino groups, gel-filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis. The amidated gelatins had higher isoionic points as expected, but their regeneration of the triple helical structure of collagen was prevented as judged from analyses of circular dichroism. Gels made from amidated gelatins showed fairly high viscosity but low gel strength.     

Nonesterified galacturonic acid sequence homology of pectins

The methyl ester distribution of pectins was studied with a recently developed enzymatic method. Endopolygalacturonase of Kluyveromyces fragilis was used to degrade pectin and the composition of the degradation products was determined with high-performance anion-exchange chromatography at pH 5. Three characteristics indicative for the distribution of nonesterified galacturonic acid residues were obtained: the percentage of nonesterified galacturonic acid residues liberated of the total number of nonesterified galacturonic acid in the undigested polymer, the proportion of nonesterified mono-, di-, and trigalacturonic acid released, and the ratio of the sum of the peak areas of methyl ester containing oligomers divided by the sum of the peak areas of the nonesterified oligomers detected. From these characteristics and the degree of methyl esterification, the mean sequence similarity of the methyl ester distributions was calculated. Computational techniques commonly employed in the determination of the sequence similarity of DNA and proteins were used to discriminate the various types of distributions found and to construct a distance tree. In general, three types of methyl ester distributions could be discerned in pectin: random, high, and blockwise esterified. This report is the first to describe a parametric approach for the comparison of the substituent distribution in polymers. The importance of this novel approach in the study of the methyl ester distribution and the functional properties of pectin is discussed.     

Interactions of amidated acids with heparin

Raman and NMR studies are performed to characterize the solution structures of complexes between heparin and a group of amidated acids, which act as delivery agents that facilitate the gastrointestinal absorption of orally administered heparin. At concentrations typically employed for the oral drug delivery of heparin, the contact points between heparin complexed with the delivery agents include points near the OH groups of heparin. The results suggest that heparin interacts rather nonspecifically with the amidated acids as monomers and with self-associated complexes of the delivery agents. It is also found that the carboxyl groups of at least one of the bioactive delivery agents easily protonates when it forms complexes with itself or heparin. This attribute may be one reason why this class of compounds is effective in the oral delivery of heparin.     

Expression, purification, and characterization of C-terminal amidated glucagon in Streptomyces lividans

Glucagon, a peptide hormone produced by alphacells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat alpha-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the Nterminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.     

Influence of the substituents of the carboxyl groups and of the rhamnose content on the solution properties and flexibility of pectins

Viscometric measurements were carried out on well-characterized apple, citrus, sugar-beet pectins in order to analyse the effect of the nature and the amount of substituents (methyl, amide, acetyl groups) and of the rhamnose content on the flexibility of the polymeric backbone. Through the dependence of the intrinsic viscosity with the ionic strength the flexibility parameter B was determined. B values between 0.072 and 0.017 indicate that pectins are relatively stiff molecules. However, an increase in flexibility is noticeable with the rise of the rhamnose content and of the amount of amide groups of the pectic acids. The flexibility is also sensitive to the degree of methylation.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

Hydrogen-ion titration curve of ovomucoid.

A systematic study of the H+ titration curve of purified ovomucoid was made at three temperatures (15, 25 and 35 degrees C) and three ionic strengths (0.05, 0.15 and 1.0). In all, 49 protons were dissociated reversibly in the pH range, 2.0-12.0. From the analysis of the results up to pH 12.0, the numbers of different dissociable groups per 28 300 g protein, together with their intrinsic pK values in parentheses were found tp be' 27 sode-chain carboxyl (pKint=4.0), four imidazole (pKint=6.5), one alpha-amino (pKint=7.5), 12 epsilon-amino (pKint=9.6), one guanidino (pKint=11.8) and one alpha-carboxyl group with abnormally low pK. The total number of basic nitrogens per mole of the protein was 22 so that four guanidino groups remained untitrated up to pH 12.0. Spectrophotometric titration showed that three out of five phenolic groups were titrated reversibly up to pH 11.9 with an intrinsic pK of 10.25; the remaining two groups became accessible only on protein denaturation. Viscosity results suggested absence of conformational change in the pH range 2.0-11.2. This explains the constancy of the pK values of carboxyl groups in the pH range 2.0-5.0. The empirical value of the electrostatic interaction factor, w, was 0.04, both in the carboxyl and phenolic regions.