Acid Gel Filtration Research Articles

Glycomacropeptide: A comprehensive understanding of its major biological characteristics and purification methodologies.

Glycomacropeptide (GMP) is a bioactive peptide derived from whey protein, consisting of 64 amino acids. It is a phenylalanine-free peptide, making it a beneficial dietary option for individuals dealing with phenylketonuria (PKU). PKU is an inherited metabolic disorder characterized by high levels of phenylalanine in the bloodstream, resulting from a deficiency of phenylalanine dehydrogenase in affected individuals. Consequently, patients with PKU require lifelong adherence to a low-phenylalanine diet, wherein a significant portion of their protein intake is typically sourced from a phenylalanine-free amino acid formula. GMP has several nutritional values, numerous bioactivity properties, and therapeutic effects in various inflammatory disorders. Despite all these features, the purification of GMP is an imperative requirement; however, there are no unique methods for achieving this goal. Traditionally, several methods have been used for GMP purification, such as thermal or acid treatment, alcoholic precipitation, ultrafiltration (UF), gel filtration, and membrane separation techniques. However, these methods have poor specificity, and the presence of large amounts of impurities can interfere with the analysis of GMP. More efficient and highly specific GMP purification methods need to be developed. In this review, we have highlighted and summarized the current research progress on the major biological features and purification methodologies associated with GMP, as well as providing an extensive overview of the recent developments in using charged UF membranes for GMP purification and the influential factors.

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Silkworm serpin32 functions as a negative-regulator in prophenoloxidase activation

The extracellular serine protease cascade is an essential component of insect humoral immunity. Serine protease inhibitors (serpins) play an important regulatory role in the process of insect immunity by regulating the serine protease cascade pathway. We aimed to clarify the function of Bmserpin32 in this study. First, we performed homologous sequence alignment and phylogenetic analysis of Bmserpin32. Bmserpin32 was found to share 64% amino acid sequence identity with Manduca sexta serpin7, an immunomodulatory protein. Bmserpin32 cDNA was cloned, and the recombinant Bmserpin32 protein was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity and gel filtration chromatography. The activity assay showed that Bmserpin32 had significant inhibitory activity against trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and site-directed mutagenesis combined with activity assays indicated that the cleavage site of Bmserpin32 is between Arg359 and Ile360. After infection with E. coli or Micrococcus luteus, the expression level of Bmserpin32 in immune-related tissues was significantly upregulated. In addition, Bmserpin32 could delay or inhibit the melanization of hemolymph by inhibiting the activation of prophenoloxidase in larval hemolymph. Furthermore, a physiological target of Bmserpin32 was identified as the clip protease, BmPAP3, an apparent ortholog of M. sexta propenoloxidase-activating protease-3. Our observations enable a better understanding of the physiological role of Bmserpin32 in regulating melanization in silkworm.

Oligosaccharide elicitor prepared from Salecan triggers the defense responses of Arabidopsis thaliana Col0 against Botrytis cinerea infection.

Oligosaccharides from the water-soluble β-glucan, Salecan, were investigated to evaluate the activation effect on the defense responses of Arabidopsis thaliana Col0. Salecan oligosaccharides (ScOs, DP 5-10) were prepared at first by acid hydrolysis and gel filtration chromatography and then employed for foliar spray on Arabidopsis seedlings and plants. After ScOs treatment, increase of hydrogen peroxide was histologically and analytically detected in leaves. Transcription levels of several indicator genes which correspond to the signaling pathways and pathogenesis-related proteins were up-regulated at different time by means of quantitative RT-PCR analysis. Importantly, the sequential activation of salicylic acid-mediated and jasmonate-mediated signaling pathway was observed in leaves. Furthermore, pot test and antimicrobial test gave the fact that pretreatment with ScOs restrained the leaf lesions caused by infection of Botrytis cinerea Bc0510 via the enhancement of defense responses of A. thaliana. In conclusion, Salecan oligosaccharides serve as an elicitor which can be used for biological control of plant pathogen.

Isolation, purification and structural characterization of a water-soluble polysaccharide HM 41 from Halenia elliptica D. Don

Abstract A water-soluble polysaccharide, HM 41 , was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM 41 was composed of rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), glucose (Glc) with a molar ratio of 1.0:5.5:1.8:3.0:9.4:21. The average molecular weight of HM 41 was approximately 1.17 × 10 4 . Periodate oxidation, Smith degradation, methylation and GC, IR, NMR, XRD, GC–MS analysis were used for the structural analysis of HM 41 . Its main chain was composed mainly of β -(1 → 4)Gal, β -(1 → 4)Glc and β -(1 → 6)Glc. β -(1 → 4)Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues; (1 → 4)Glc had no branch; (1 → 6)Glc were substituted at 3- O and on average there were 9 branches among 14 main chain residues. The side chain was composed of (1 → 3,6)-Rha, (1 → 4)/(1 → 5)-Ara, (1 → 4)/(1 → 5)-Xyl, (1 → 4,6)-Man and (1 → 2)-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM 41 had strong scavenging activities in vitro hydroxyl. Overall, HM and HM 41 may have potential applications in the antioxidants for medical and food industry.

Isolation, Purification and Identification of Parvalbumin Allergen in <em>Cyprinus carpio</em>

Parvalbumin (PV) is a major fish allergen that is characterized by high water solubility, low molecular weight (i.e., ~12,000), acidic isoelectric point, heat stability and high phenylalanine content (compared with tyrosine and tryptophan). We used a fast, simple and efficient method to purify PV from <em>Cyprinus carpio</em> (carp). Heating, two runs of trichloroacetic acid precipitation and gel filtration chromatography were integrated in this method. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot showed that the purified protein was PV and the purity of carp PV obtained using this method was more than 90%. Moreover, the PV was characterized by MALDI-TOF-TOF MS and an unusually high 260 to 280 nm run absorbance ratio in the final purification stages. The rapid purification method used in this study resulted in better purification and identification of PV compared with other methods. Further studies on PV detection in freshwater fish can be performed based on the results of this experiment.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance.

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 5 s −1 and 4.7 × 10 6 s −1 .M −1 , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg 2+ , Cd 2+ , K + and Ca 2+ , and it was drastically inhibited by F − . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

Isolation and Characterization of Edestin from Cheungsam Hempseed

Edestin, a major hempseed storage protein from Cheungsam, was isolated to apparent homogeneity by acid precipitation and gel filtration chromatography. The native molecular weight of purified edestin was approximately 300 kDa by Sephacryl S-300 gel filtration. Upon adding 2-mercaptoethanol, the isolated edestin of 56.7 kDa on the non-reduced sodium dodecyl sulfate polyacrylamide gel was converted into subunits, suggesting that the protein might be composed of subunits linked by disulfide bond. Cheungsam edestin was rich in essential amino acids and it has 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity. The results suggest that Cheungsam edestin might be utilized as a superior antioxidative nutrient.

Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

Purification and primary structure of glucagon from ostrich pancreas splenic lobes

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase.

BackgroundThe emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.ResultsIn the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.ConclusionThis is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.

Soluble and glyco-lipid modified baculovirus Plasmodium falciparum C-terminal merozoite surface protein 1, two forms of a leading malaria vaccine candidate

Recombinant homologues of the Plasmodium merozoite surface protein 1 C-terminus are leading blood stage malaria vaccine candidates. MSP1 is anchored to the merozoite plasma membrane in vivo by a glycosyl-phosphatidyl-inositol (GPI) moiety, implicated in malaria pathology. Two types of recombinant Plasmodium falciparum MSP1p19 (PfMSP1p19) expressed in baculovirus/insect cells are described here: (1) a soluble, secreted form (PfMSP1p19S) and (2) detergent soluble cellular form(s) (PfMSP1p19 + A), released from the infected cell surface by treatment with GPI specific phosphatidyl-inositol phospholipase C (PI-PLC). Soluble and cellular PfMSP1p19 were purified and characterized using SDS-PAGE, mass spectrometry (MS), N-terminal amino acid sequencing, gel filtration and glycan analyses. Quantitative inositol dosage suggested that surface GPI processed entities constituted only 14% of the purified cellular PfMSP1p19 + A, with GPI unprocessed forms likely recovered in the endoplasmic reticulum. Nevertheless, this preparation has dramatic immuno-stimulatory activity to be described elsewhere. The interest of these results for both malaria specific and generic vaccine development are discussed.

Study of the Antifungal Ability of &amp;lt;italic&amp;gt;Bacillus subtilis&amp;lt;/italic&amp;gt; Strain PY-1 &amp;lt;italic&amp;gt;in Vitro&amp;lt;/italic&amp;gt; and Identification of its Antifungal Substance (Iturin A)

A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-betaAA-Asn-CO+, was first reported.

Application of response surface methodology to optimize production of poly-ɛ-lysine by Streptomyces albulus IFO 14147

Response surface methodology (RSM) were employed to study the effect of yeast extract, glucose, ammonium sulfate, and initial medium pH on the production of poly-ɛ-lysine (ɛ-PL) by Streptomyces albulus IFO 14147 in shaken flask cultures. The results of first-order factorial design experiments showed that the linear terms of yeast extract, glucose, and ammonium sulfate had significant effect on ɛ-PL production, but the initial medium pH at the tested range exhibited insignificant effect on ɛ-PL production. It was predicted that decreasing the concentration of yeast extract, glucose, and ammonium sulfate enhance ɛ-PL production. Furthermore, the results of the four-factor-two-level (2 4) factorial experiments indicated that the first order with interactions model ( R 2 value of 0.9660) appeared to be more adequate than the pure first-order model ( R 2 value of 0.8759). The optimum composition was then assessed by using a central composite design (CCD). The experimental results of CCD were fitted with a second-order polynomial equation by a multiple regression analysis. The determination coefficient R 2 was 0.816, the Fisher F-test was significant at upper 5% level and the lack of fit was insignificant at 5% level. All of these indicated a good adequacy of the second-order polynomial model proposed to explain the data observed. The optimal ɛ-PL yield (8.90 ± 2.14 g/L) determined by the CCD was predicted at the regions where concentrations of yeast extract, glucose, and ammonium sulfate were at 2.86, 23.82, and 0.14 g/L, respectively. Verification of the calculated optimum was done with a culture medium representing this optimal point and yielding 8.13 ± 1.87 g/L (average of three repeats). The yield of ɛ-PL by S. albulus IFO 14147 was increased significantly by 984%, from 0.75 to 8.13 g/L when the strain was cultivated in the optimal medium developed by surface response methodology, as compared to conventional medium M3G used in the literature. The ɛ-PL thus produced was well characterized by chemical tests, amino acid analysis and gel filtration technique.

Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Isoamylase catalyzes the hydrolysis of α-1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279 … 11435) encoding the thermophilic isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 °C. They had an apparent optimal pH of 5 and an optimal temperature at 75 °C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 °C for the wild-type and His-tagged enzymes, respectively. These thermophilic isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature.

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Lithium [Bis{benzoyl‐W(CO)5}(μ2‐hydrogen)] − A Charge‐Assisted H+‐Bridged Organometallic Complex Prepared by Selective Li+ Ion Replacement

AbstractEfficient syntheses and isolation procedures of two closely related benzoyl−W(CO)5 compounds, [{Li(diglyme)}{Ph(O)CW(CO)5}] (1) and [Li(diglyme)2][(μ2‐H){Ph(O)CW(CO)5}2] (2) are reported. The empirical formulae of 1 and 2 differ only in that the latter contains half the number of Li+ ions, with the balance of cations replaced by protons through an acidic silica gel filtration procedure. X‐ray structural investigations of both compounds revealed a self‐assembled polymeric Li+‐bridged structure for 1, and a strikingly dissimilar H+‐bridged structure for 2, thereby emphasising the important influences of strong, charge‐assisted hydrogen bonds on solid‐state structures. Possible implications of this novel route to charge‐assisted hydrogen bonds for crystal engineering are discussed. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2003)

ISOLATION AND PARTIAL CHARACTERIZATION OF CATALYTIC ANTIBODIES WITH OLIGONUCLEASE ACTIVITY FROM BOVINE COLOSTRUM

ABSTRACT Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA fromEscherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75°C for 52 min.