Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.     

鹰嘴豆β-半乳糖苷酶的分离纯化与表征

从鹰嘴豆中分离得到了三种β－半乳糖苷酶（酶Ⅰ、酶Ⅱ和酶Ⅲ）。将酶Ⅰ和酶Ⅱ进一步纯化,其比活力分别提高了19倍和48倍．酶活力回收率分别为16％和18％,测得它们的表观分子里分别为2．4×104和5.8×104;最适pH分别5.9和5.0,最适温度分别为55℃和45℃．酶Ⅰ水解ONPG和PNPG的KM分别为33×10-2mol·dm-3和60×10-3mol·dm－3;酶Ⅱ水解ONPG和PNPG的KM分别为30×10－3mol·dm－3和60×10－4mol·dm－3.乳糖和半乳糖为该酶的竞争性可逆抑制剂,棉子糖为非竞争性可逆抑制剂．该酶受Hg2＋和PCMB强烈抑制和NEM明显抑制,而Mg2＋、Zn2＋和Ca2＋具有激活作用,推知巯基（－SH)是酶活性中心必须基团．     

pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Characterization of aldose reductase and aldehyde reductase from the medulla of rat kidney

Aldose reductase and aldehyde reductase from the medulla of the rat kidney have been purified to homogeneity by using affinity chromatography, gel filtration and chromatofocusing. The molecular weights of aldose reductase and aldehyde reductase by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were found to be 37000 and 39000, respectively. The isoelectric points of aldose reductase and aldehyde reductase were found to be 5.4 and 6.2 by chromatofocusing, respectively. The major differences of amino acid compositions between both enzymes were found in serine, alanine and aspartic acid. Substrate specificity studies showed that aldose reductase utilized aldo-sugars such as D-glucose and D-galactose, but aldehyde reductase did not use them. The Km values of aldose reductase for various substrates were lower than those of aldehyde reductase. Aldose reductase utilized both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, whereas aldehyde reductase utilized only NADPH. The presence of the sulfate ion resulted in a dramatic activation of aldose reductase whereas it did not affect aldehyde reductase activity. These enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldose reductase was more susceptible than aldehyde reductase to inhibition by the aldose reductase inhibitors.     

Reverse-phase HPLC separation of D-amygdalin and neoamygdalin and optimum conditions for inhibition of racemization of amygdalin

In boiling aqueous solution, D-amygdalin usually begins to convert into neoamygdalin in 3 min and more than 30% of the initial D-amygdalin is found as neoamygdalin after 30 min. In this report, we establish methods for simple HPLC analysis and the inhibition of D-amygdalin conversion. D-Amygdalin and its conversion product, neoamygdalin, were clearly separated on reverse-phase column chromatography by an optimized eluent of 10 mM sodium phosphate buffer (pH 3.8) containing 6% acetonitrile. Linearity for analyzing D-amygdalin and neoamygdalin was observed in the range from 0.05 to 0.5 mM. The detection limits for D-amygdalin and neoamygdalin were ca. 5 microM per injected amount. We found that D-amygdalin conversion was completely inhibited by adding 0.05% citric acid to the aqueous solution before boiling. To prevent the loss of pharmaceutical potency of Tonin, we applied this method to measure the conversion rate of D-amygdalin. We confirmed that D-amygdalin conversion in Tonin is effectively inhibited by acidic boiling solution with 0.1% citric acid.     

Presence of protein polymorphism in karasurin, an abortifacient and anti-tumor protein, identified with physicochemical properties.

Karasurin is an abortifacient plant protein isolated from fresh root tubers of Trichosanthes kirilowii Maximowicz var. japonicum Kitamrra (Cucurbitaceae). This study describes the presence of protein polymorphism in karasurin (karasurin-A and karasurin-B) separated by ion-exchange chromatography. Two components showed no differences in the molecular weight (ca. 28000), the amino acid composition, the neutral sugar content and part of the amino acid sequence. A unique difference was observed in their isoelectric points (10.1 for karasurin-A and 10.2 for karasurin-B). However, modifications of glycosylation or phosphorylation were not found. Biological assays for inducing mid-term abortion in pregnant mice and inhibition of the growth of BeWo cells gave no significant differences between them. The presence of protein polymorphism in karasurin, whose biological significance is not yet understood, is a first finding among several abortifacient proteins.     

Copper(II) interaction with prion peptide fragments encompassing histidine residues within and outside the octarepeat domain: speciation, stability constants and binding details

A 31-mer polypeptide, which encompasses residues 84-114 of human prion protein HuPrP(84-114) and contains three histidyl residues, namely one from the octarepeat (His85) and two histidyl residues from outside the octarepeat region (His96 and His111), and its mutants with two histidyl residues HuPrP(84-114)His85Ala, HuPrP(84-114) His96Ala, HuPrP(84-114)His111Ala and HuPrP(91-115) have been synthesised and their Cu2+ complexes studied by potentiometric and spectroscopic (UV/Vis, CD, EPR, ESI-MS) techniques. The results revealed a high Cu2+-binding affinity of all peptides, and the spectroscopic studies made it possible to clarify the coordination mode of the peptides in the different complex species. The imidazole nitrogen donor atoms of histidyl residues are the exclusive metal-binding sites below pH 5.5, and they have a preference for macrochelate structure formation. The deprotonation and metal-ion coordination of amide functions take place by increasing the pH; all of the histidines can be considered to be independent metal-binding sites in these species. As a consequence, di- and trinuclear complexes can be present even in equimolar samples of the metal ion and peptides, but the ratios of polynuclear species do not exceed the statistically expected ones; this excludes the possibility of cooperative Cu2+ binding. The species with a (N(im),N,N)-binding mode are favoured around pH 7, and their stability is enhanced by the macrochelation from another histidyl residue in the mononuclear complexes. The independence of the histidyl sites results in the existence of coordination isomers and the preference for metal binding follows the order of: His111>His96>His85. Deprotonation and metal-ion coordination of the third amide functions were detected in slightly alkaline solutions at each of the metal-binding sites; all had a (N(im),N,N,N)-coordination mode. Spectroscopic measurements also made it clear that the four lysyl amino groups of the peptides are not metal-binding sites in any cases.     

Vitamin K2 as a New Modulator of the Ceramide De Novo Synthesis Pathway

The aim of the study was to evaluate the influence of vitamin K2 (VK2) supplementation on the sphingolipid metabolism pathway in palmitate-induced insulin resistant hepatocytes. The study was carried out on human hepatocellular carcinoma cells (HepG2) incubated with VK2 and/or palmitic acid (PA). The concentrations of sphingolipids were measured by high-performance liquid chromatography. The expression of enzymes from the sphingolipid pathway was assessed by Western blotting. The same technique was used in order to determine changes in the expression of the proteins from the insulin signaling pathway in the cells. Simultaneous incubation of HepG2 cells with palmitate and VK2 elevated accumulation of sphinganine and ceramide with increased expression of enzymes from the ceramide de novo synthesis pathway. HepG2 treatment with palmitate and VK2 significantly decreased the insulin-stimulated expression ratio of insulin signaling proteins. Moreover, we observed that the presence of PA w VK2 increased fatty acid transport protein 2 expression. Our study showed that VK2 activated the ceramide de novo synthesis pathway, which was confirmed by the increase in enzymes expression. VK2 also intensified fatty acid uptake, ensuring substrates for sphingolipid synthesis through the de novo pathway. Furthermore, increased concentration of sphingolipids, mainly sphinganine, inhibited insulin pathway proteins phosphorylation, increasing insulin resistance development.     

Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding.     

In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α-amylase and α-glucosidase

This work reports the in vitro inhibitory activity of water decoctions of leaves, germ flour, pulp, locust bean gum and stem bark of carob tree on α-amylase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase. The antioxidant activity and the chemical characterisation of the extracts made by spectrophotometric assays and by high-performance liquid chromatography are also reported. Leaves and stem bark decoctions strongly inhibited all the enzymes tested, had significant antioxidant activity and the highest total phenolics content. The major compounds were identified as gallic acid in the leaves and gentisic acid in the stem bark.     

Effects of interaction of tannins with co-existing substances. VII. Inhibitory effects of tannins and related polyphenols on xanthine oxidase

The inhibitory effects of hydrolyzable tannins, condensed tannins and related polyphenols on the activity of xanthine oxidase (XOD), catalyzing uric acid formation from xanthine, were investigated. Marked differences in the strength of the inhibition were observed. Some of the differences among the monomeric hydrolyzable tannins were due to their molecular weights, reflecting the number of phenolic hydroxyl groups in the molecule. However, the inhibitory activity of several oligomeric hydrolyzable tannins seemed particularly low in spite of their large molecular size. It was also observed that differences in location of acyl groups on the carbohydrate cores caused differences in the inhibitory activity among monomeric and oligomeric hydrolyzable tannins. A caffeic acid derivative (caffeetannin), 3,5-di-O-caffeoylquinic acid (24), also inhibited this enzyme. Galloylation and the degree of polymerization in proanthocyanidins were also shown to affect remarkably the strength of the inhibition. Among the compounds tested in the present study, valoneic acid dilactone (29), isolated from Mallotus japonicus, inhibited the enzyme most effectively. A kinetic study showed that this dilactone inhibited XOD non-competitively. Comparison of the inhibitory effect on XOD, with the binding activity to hemoglobin, for each tannin, suggests that their inhibition of XOD is not based on non-specific binding to the protein. Similar comparison of the inhibitory effect on XOD with the inhibitory effect on the generation of superoxide anion radical (O2-.) from the hypoxanthine-XOD system revealed that the inhibition of O2-. generation by tannins is due to their radical-scavenging activity, and not due to their inhibitory activity upon the enzyme.     

Le blocage de la lysine par la réaction de MAILLARD. I. Synthèse de N-(désoxy-1-D-fructosyl-1)- et N-(désoxy-1-D-lactulosyl-1)-L-lysines

The authors describe a specific method for the preparation of N?-(1-deoxy-D-fructosyl)-L-lysine, Nα-(1-deoxy-D-fructosyl)-L-lysine, their formyl derivatives, and Nα-formyl-?-(1-deoxy-D-lactulosyl)-L-lysine, as well as Nα, N?-di-(1-deoxy-D-fructosyl)-L-lysine. After purification by cation-exchange chromatography using volatile pyridine-acetic acid or pyridine-formic acid buffers, these compounds are obtained in the pure state. By thin-layer chromatography and paper electrophoresis these lysine derivatives can be rapidly separated and identified.     

Reversed-phase high-performance liquid chromatographic characterization of acetic acid extracts of the normal and the diabetic human pancreas

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. greater than 6000 dalton (Peak I) or with mol. wt. less than or equal to 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscured chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifluoroacetic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of sialuria by cation-exchange high-performance liquid chromatography

A simple and selective method for the detection of sialuria by high-performance liquid chromatography is described. The urine sample (2 ml) is purified using a C18 cartridge or ion-exchange chromatography, and free N-acetylneuraminic acid is separated on an Aminex HPX-87 cation-exchange column using 3 mM sulphuric acid as the mobile phase. The retention time of N-acetylneuraminic acid is ca. 8 min and the detection limit ca. 1 mumol/l. The within-day coefficient of variation is less than 4.9% and the day-to-day coefficient of variation is less than 5.6%. The method was tested on twenty normal individuals and four sialuria patients.     

Adsorption Study of Immunoglobulin G Subclasses from Different Species by Pseudobioaffinity Separation on Histidyl–Bisoxirane–Sepharose

Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG₁, IgG₂, and IgG₃, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.     

Autoxidation of ascorbic acid catalyzed by the copper(II) bound to L-histidine oligopeptides, (His)iGly and acetyl-(His)i Gly (i=9, 19, 29). Relationship between catalytic activity and coordination mode

Spectroscopic and kinetic studies on the autoxidation of ascorbic acid catalyzed by copper complexes of histidine oligopeptides, (His)iGly (i=4, 9, 19, 29), and their acetyl derivatives, Ac-(His)iGly (i=9, 19) have been carried out at pH 4.4 and 25 degrees C under dioxygen. The reaction was monitored at 260 nm using a stopped-flow spectrophotometric technique. The reaction fitted the "Michaelis- Menten" mechanism, and ascorbate was oxidized by the "Ping-Pong" mechanism. The Cu(lI) complexed with the oligopeptide (i > or = 9) enhanced the reaction approximately two-fold relative to the aqueous Cu(II). The catalytic activity depends on the molecular weight which is related to the number of histidyl residues and on the coordination mode of the copper-binding site. Results of circular dichroism (CD) experiments revealed the existence of two types of Cu(II). The catalytically active Cu(II), which is accommodated in the imidazole clusters composed of at least six histidyl residues, exhibits d-d transition bands at 520 and 630 nm, and is easily dissociable, enhances the autoxidation; Ac-(His)19Gly is likely to accommodate approximately three active Cu(II) ions. The Cu(II), which is complexed tightly with the terminal H2N-X-Y-His- moiety, where X and Y denote amino acids, inhibits the autoxidation, and exhibits absorption bands at 480 and 550 nm.     

Adsorption Study of Immunoglobulin G Subclasses from Different Species by Pseudobioaffinity Separation on Histidyl–Bisoxirane–Sepharose

Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG₁, IgG₂, and IgG₃, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.

     

A rapid assay for angiotensin‐converting enzyme activity using ultra‐performance liquid chromatography–mass spectrometry

Angiotensin‐converting enzyme (ACE) plays an important role in the renin–angiotensin system and ACE activity is usually assayed in vitro by monitoring the transformation from a substrate to the product catalyzed by ACE. A rapid and sensitive analysis method or ACE activity by quantifying simultaneously the substrate hippuryl–histidyl–leucine and its product hippuric acid using an ultra‐performance liquid chromatography coupled with electrospray ionization‐mass spectrometry (UPLC‐MS) was first developed and applied to assay the inhibitory activities against ACE of several natural phenolic compounds. The established UPLC‐MS method showed obvious advantages over the conventional HPLC analysis in shortened running time (3.5 min), lower limit of detection (5 pg) and limit of quantification (18 pg), and high selectivity aided by MS detection in selected ion monitoring (SIM) mode. Among the six natural products screened, five compounds, caffeic acid, caffeoyl acetate, ferulic acid, chlorogenic acid and resveratrol indicated potent in vitro ACE inhibitory activity with IC₅₀ values of 2.527 ± 0.032, 3.129 ± 0.016, 10.898 ± 0.430, 15.076 ± 1.211 and 6.359 ± 0.086 mm , respectively. A structure–activity relationship estimation suggested that the number and the situation of the hydroxyls on the benzene rings and the acrylic acid groups may play the most predominant role in their ACE inhibitory activity. Copyright © 2009 John Wiley & Sons, Ltd.