Rigorous Purification Research Articles

Effects of the Tibetan herbal preparation PADMA 28 on blood lipids and lipid oxidisability in subjects with mild hypercholesterolaemia

Previous studies showed an anti-atherosclerotic effect of PADMA 28, an herbal formula based on Tibetan medicine. As the mechanisms of action are not fully understood, we investigated whether PADMA 28 may lower blood lipids and lipid oxidisability, and affect early endothelial dysfunction. Sixty otherwise healthy subjects with total cholesterol > or = 5.2 mmol/l and < 8.0 mmol/l were randomly assigned to placebo or PADMA 28, 3 x 2 capsules daily, for 4 weeks (double-blind). Blood lipids (total, LDL-, and HDL-cholesterol, triglycerides, Apo-lipoprotein A1 and B) and ex vivo lipid oxidisability were measured before and after treatment. In a subset of 24 subjects, endothelial function was assessed using venous occlusion plethysmography with intraarterial infusion of acetylcholine. Isolated LDL and plasma both untreated and pre-treated with PADMA 28 extract were oxidised by the radical generator AAPH. Conjugated diene formation was measured at 245 nm. Blood lipids did not change during the study in both groups. In contrast to previous reports in mild hypercholesterolaemia, no endothelial dysfunction was seen and, consequently, was not influenced by therapy. Ex vivo blood lipid oxidisability was significantly reduced with PADMA 28 (area undercurve: 5.29 +/- 1.62 to 4.99 +/- 1.46, p = 0.01), and remained unchanged in the placebo group (5.33 +/- 1.88 to 5.18 +/- 1.78, p > 0. 1). This effect persisted one week after cessation of medication. In vitro experiments confirmed the prevention of lipid peroxidation in the presence of PADMA 28 extracts. Persistent protection was also seen for LDL isolated from PADMA 28-pretreated blood after being subjected to rigorous purification. This study suggests that the inhibition of blood lipid oxidisability by PADMA 28 may play a role in its anti-atherosclerotic effect.

Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins

Patients with psoriasis, a chronic, hyperproliferative and noninfectious skin disease, suffer surprisingly fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides psoriatic skin with resistance against infection. We subsequently began a systematic analysis of in vitro antimicrobially active proteins in psoriatic-scale extracts. A biochemical approach with rigorous purification and characterization combined with antimicrobial testing identified a number of mostly new human antibiotic peptides and proteins. In this review, we will focus on the most prominent antimicrobial proteins in psoriatic-scale extracts, which we identified as the S100-protein psoriasin, human beta-defensin 2 (hBD-2), RNase 7, lysozyme, and human neutrophil defensin 1-3. Apart from these cutaneous, antimicrobial proteins, only a few others, including hBD-3, have been characterized. A great number of minor antimicrobial proteins await further structural characterization.

Investigation of the solid phase synthesis of tyrosine‐derived diphenol monomers with resin‐bound carbodiimide coupling reagents

AbstractTyrosine‐derived pseudo‐polypeptides have recently been established as potential biomaterials. The bulk production of these polymers is dependant upon the convenient synthesis of tyrosine‐derived diphenolic monomers. Suitable solution phase methods for the synthesis of such monomers, with transient activated esters, have been reported previously by Kohn, J.; Langer, R. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E., Eds.; Academic Press: New York, 1996; pp 64–72. However, for all the methods reported by them, purification and isolation of the diphenol monomer involves rigorous extraction, column purification, or an extensive aqueous workup. These may lead to the incorporation of solvent‐based as well as inorganic impurities and also may lead to difficulties in the process scale‐up. In this article, an alternate and relatively more convenient method for the synthesis of tyrosine‐based diphenol monomers is reported. This involved the investigation of polymer resin‐bound carbodiimide in the solid‐phase synthesis of L‐tyrosine‐based diphenolic monomers. This method was found to eliminate the need for rigorous purification processes and was found to maintain reasonable yield as well as maintain purity of the final monomer product. The monomer was able to produce polymers of a reasonably high‐molecular‐weight and a narrow polydispersity. The amide bond formation by such a polymer‐tethered reagent can be described to follow a reverse‐Merrifield sense and the method is relatively convenient to scale up. The L‐tyrosine‐based diphenolic monomeric compound formed by this method was analyzed by NMR, Fourier transform infrared, and elemental microanalysis techniques for chemical structure and composition. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4906–4915, 2004

Interaction-controlled HPLC for block copolymer analysis and separation.

An interaction-controlled HPLC technique has been developed to analyze homopolymer precursors in block copolymer systems that are not easily identified by size exclusion chromatography (SEC) and to obtain block copolymers that are homopolymer-free and compositionally narrower than the as-synthesized ones. We demonstrate that a "single peak" in SEC does not necessarily mean that the block copolymers are free of homopolymers (due to limitations in the SEC analysis of block copolymers) and propose to employ the interaction-controlled HPLC strategy for rigorous analysis and purification of block copolymers in terms of their chemical heterogeneity.

Emulsion Polymerization of Vinyl neo-Decanoate, a “Water-Insoluble” Monomer

Rate and particle size data for the emulsion polymerization of vinyl neo-decanoate, where inhibitor/retarder effects are obviated through rigorous purification, can be quantitatively fitted by conventional mechanisms for particle formation and growth, even though the monomer has a very low water solubility. These mechanisms are (1) radical entry is by an oligomeric radical (z-mer) arising from aqueous-phase propagation at a critical degree of polymerization (z between I and 2 for this monomer) where the species becomes surface-active, (2) radical loss is by transfer to a species which desorbs into the aqueous phase and eventually terminates' (after reentry into another particle) rather than undergoing further propagation, and (3) particle formation is by a z-mer entering a micelle. These hypotheses are consistent with gamma relaxation data which give direct information on radical loss.

Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands.

This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

Reconstitution of human DNA polymerase delta using recombinant baculoviruses: the p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme.

Eukaryotic DNA polymerase delta is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase delta. However, rigorous purification of human or bovine DNA polymerase delta from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase delta, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase delta displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase delta efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase delta.

New evidence that the Alzheimer β-amyloid peptide does not spontaneously form free radicals: An ESR study using a series of spin-traps

The direct formation of free radicals from Aβ has been suggested to be a key neurotoxic mechanism in Alzheimer’s disease (AD). We have explored the possibility of the spontaneous formation of peptide-derived free radicals during the incubation of Aβ 1-40 by ESR spectroscopy using N-tert-butyl-α-phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt (DBNBS) as spin traps. Employing PBN, we observed spectra during the incubation of β-amyloid peptide, at 37°C, which included adducts of 2-methyl-2-nitrosopropane (MNP), despite rigorous purification of the PBN before incubation. The formation of some of these adducts was found to be enhanced by ambient laboratory light. Our experiments have led us to propose a hypothesis that PBN undergoes hydrolysis and decomposition in the presence of oxidants, which explains the origin of all of the PBN and MNP adducts observed (even when the PBN is highly purified). Hydrogen peroxide, formed during incubation, could play a major role as an oxidant in these experiments. Of the other three spin traps, only DMPO gave (very weak) spectra, but these could be assigned to its hydroxyl radical adduct, formed as an artifact by the nucleophilic addition of water to DMPO, catalyzed by trace levels of iron ions. Thus, while spectra are observed during our experiments, none of them can be assigned to adducts of radicals derived from the peptide and, therefore, our data do not support the suggestion that radicals are spontaneously formed from β-amyloid peptide.

Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1.

The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both alpha-(1,4)- and alpha-(1,6)-glycosidic linkages of different linear and branched polysaccharides. The following observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes. Even though the enzymes were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found to migrate in a single peak. By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE, and zymographic analysis on different polysaccharides. Preferential chemical modification of the enzyme and concomitant high-performance thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl carbodiimide-HCl and cleaved alpha-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose. On the other hand, formalin-sensitive neopullulanase acts on both alpha-(1,4) and alpha-(1,6) linkages of pullulan and starch with maltotriose and panose as major products. It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase toward the hydrolysis of alpha-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not seen in zymograms, in which the enzymes are physically separated during electrophoresis. It is presumed that SDS-protein intercalation dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost. The optimum temperature and pH of amylase and neopullulanase were 60 degreesC and 8.0, respectively. The enzymes were found stable in high alkaline pH for 24 h. Therefore, the saccharifying alkaline amylase and neopullulanase of M. halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features.

Reductive metabolism of Cr(VI) by cysteine leads to the formation of binary and ternary Cr--DNA adducts in the absence of oxidative DNA damage.

Carcinogenic chromium(VI) compounds require reduction for the induction of genotoxicity. In this work, we examined a spectrum of DNA damage produced in Cr(VI)-cysteine reactions at neutral pH. Cr(VI) reduction followed single-component kinetics and led to a significant oxidation of 2',7'-dichlorofluoroscein (DCFH). The presence of residual Fe and/or Cu resulted in an increased level of oxidation of DCFH, and the removal of adventitious metals required rigorous purification of cysteine. DNA breakage and abasic sites were not detected, suggesting that DNA is much less susceptible to oxidation than DCFH. Cr(VI) reduction led to the extensive formation of Cr-DNA adducts and Cys-Cr-DNA and interstrand DNA-DNA cross-links. Cr-DNA binding resulted in unwinding of supercoiled DNA and a greater stability of the DNA duplex to denaturation. Ionically bound Cr comprised 40-60% of the total DNA-bound Cr, while the remaining Cr-DNA complexes represented stable Cr-DNA adducts that exhibited significant resistance to dissociation by EDTA. The yield of Cr-DNA adducts was strongly influenced by the nature of the buffer that was used. Phosphate buffer completely blocked Cr-DNA binding, whereas adduct formation in organic buffers was largely dependent on the extent of buffer ionization. The level of formation of Cr-DNA adducts was several times higher at pH 6 which resulted from lower levels of buffer ionization and diminished competition from hydroxyl ions. Yield of a number of Cr-DNA and Cys-DNA adducts increased linearly as a function of Cr(VI) concentration, whereas formation of interstrand DNA cross-links exhibited exponential dose dependence. Approximately 60-90 min was required to convert a Cr-DNA monoadduct into a DNA-DNA cross-link. Prolonged incubations at alkaline conditions led to a selective cleavage of cross-linked DNA. The alkali sensitivity of Cr-adducted DNA suggests that results of alkaline elution analysis of DNA damage in cells require a more cautious interpretation. Overall, a spectrum of DNA damage derived from Cr(VI)-cysteine reactions was similar to that found in exposed cells.

The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity.

Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. To study whether Fel d 1 is associated with enzyme activity. Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.

T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates.

The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5' end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5' end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5' ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5' end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5' end heterogeneity in combination with the 3' end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

Synthesis and characterization of arylene ether imide reactive oligomers and polymers containing diaryl alkyl phosphine oxide groups

The diamine monomer bis(m-aminophenyl) methyl phosphine oxide (DAMPO) was synthesized via nitration and reduction of diphenyl methyl phosphine oxide. Rigorous purification of this monomer enabled its utilization in the synthesis of high molecular weight poly(ether imide)s. Both thermoplastic materials and thermosetting systems, endcapped with either phthalic or phenylethynylphthalic anhydride, respectively, have been produced. Major emphasis has been placed on polyimides derived from 2,2′-bis(4-(3,4-dicarboxyphenoxy) phenyl) propane dian- hydride, also known as bisphenol-A dianhydride, or BPADA. High molecular weight homo- and copolyimides based on BPADA/DAMPO had glass transition temperature values in the range of 215–223°C, and were totally amorphous. They displayed higher modulus and tensile strength values than the polyetherimide control based on meta-phenylene diamine and also generated high TGA char yields in air. Phenylethynyl crosslinkable materials were effectively cured at 380°C to produce networks that are ductile, very solvent resistant and also generate high char yields, which suggest their possible utilization in fire resistant matrix systems. © 1998 John Wiley & Sons, Ltd.

Synthesis and Biological Activity of Metabolites of Environmental Nitroarenes

Abstract Nitroarenes are released into the environment from combustion processes; many compounds of this class are potent mutagens, and some may be carcinogens. The oxygenated derivatives of nitroarenes may be formed through atmospheric transformations of nitroarenes; as metabolites of the nitroarenes in biological systems these may be the species that mediate biological effects in vivo. We describe here efficient and convenient routes for preparation of individual nitropyrenol, acetamidopyrenol, nitrofluoranthenol and acetamidofluoranthenol isomers in high purity and good yields, and a comparison of their potency in the Ames plate incorporation assay. The synthetic approach was based on functionalization of the aromatic nucleus by acetylation or benzoylation, directed where necessary by selective desaturation of the aromatic moiety, followed by conversion to the corresponding ester and nitration. Rigorous purification was carried out before or after hydrolysis of the ester, based on the ease of separation of the esters compared to the phenols. Pyrene derivatives substituted at the 1 and 6 positions and the 1 and K-region positions were strongly mutagenic, while hydroxylation at other positions on the aromatic periphery decreased activity. 3-Nitrofluoranthen-8-ol was the most potent of the hydroxylated fluoranthene derivatives.

Overexpression, isolation, and crystallization of proteins.

AbstractRapid developments in recombinant technology have made it possible to overproduce selected proteins of specific interest to the levels required for structural analysis by X-ray crystallography. High-level gene expression has facilitated the purification of many proteins that are normally only expressed at low concentrations, as well as those that have proven difficult to purify to homogeneity from natural sources. Furthermore, advances in oligonucleotide site-directed mutagenesis have enabled proteins to be engineered so as to possess certain features that may confer stability or assist in then isolation. There are several examples of proteins that, despite rigorous purification from their natural source, have defied crystallization attempts, e.g., human growth hormone, but have been successfully crystallized from recombinant sources (1). The lack of posttranslational processing in bacterial expressed proteins can often be an advantage to the crystallographer where microheterogeneity presents a problem. Indeed, certain features or residues of a protein that are believed to impede crystal formation by preventing a close-packing arrangement may be successfully deleted by genetic manipulation without destroying its essential functionality (2).KeywordsRibosome Binding SiteLarge Single CrystalInsoluble Inclusion BodyPlasmid InstabilityPrepacked ColumnThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Evidence that thyroglobulin has an associated protein kinase activity correlated with the presence of an adenosine triphosphate binding site

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo-labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.

Evidence that thyroglobulin has an associated protein kinase activity correlated with the presence of an adenosine triphosphate binding site.

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo-labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.

FR66979 requires reductive activation to cross-link DNA efficiently

The activity of FR66979 as a DNA-DNA interstrand cross-linking agent in the absence of exogenous reducing agents was re-examined. Samples of FR66979 prepared by hydrogenation of FR900482 (H 2/Pd-C) were quite active whereas reduction of FR900482 with sodium borohydride afforded inactive or very weakly active FR66979 samples. Rigorous purification of FR66979 from either source abolished the observed cross-linking activity thus indicating that FR66979 does not efficiently cross-link DNA in the absence of exogenous reducing agents. LC-MS analysis of active samples indicated the possible presence of the over-reduced impurity 3, below. The DNA-DNA cross-link arising from the same sample was identical in structure to that formed by reductively activated FR66979.

Desulfoviridin, a multimeric-dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies.

Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady-state kinetic parameters have been determined: kcat(SO3(2-) = 0.31 mol SO3(2-).s-1.mol heme-1, Km = 0.06 mM; kcat(NO2-) = 0.038 mol NO2-.s-1.mol heme-1, Km = 0.028 mM; kcat(NH2OH) = 29 mol NH2OH.s-1.mol heme-1, Km = 48 mM. A detailed comparison is made with the Escherichia coli and spinach assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.

Jewish Lore in Manichaean Cosmogony. Studies in the 'Book of Giants' Traditions

A work entitled the of figures in every list of the Manichaean canon preserved from antiquity. Both the nature of this work and the intellectual baggage of the third-century Persian prophet to whom it is ascribed remained unknown to scholars until 1943, when fragments of several Middle Iranian versions of the Book of Giants were published by W. B. Henning. Twenty-eight years later, J. T. Milik discovered several copies of a fragmentary Aramaic work at Qumran which is unquestionably the precursor of the later Manichaean recension. One other important work, Mani's autobiography, the so-called Cologne Mani Codex, was brought to scholarly attention in 1970 with evidence that Mani spent his youth among the Elchasaites, a Judeo-Christian sect that observed the Sabbath, strict dietary laws, and rigorous purification practices. Although leading Orientalists of the nineteenth and twentieth centuries have consistently stressed the Iranian component in Mani's thought, Reeves argues, in the light of evidence drawn from the above-mentioned discoveries and from a rich panorama of other textual sources, that the fundamental structure of Manichaean cosmogony is ultimately indebted to Jewish exegetical expansions of Genesis 6:1-4. Reeves begins with an examination of the ancient testimonies about the contents of Mani's Book of Giants. Then, using documents from Second Temple Judaism, classical Gnostic literature, Christian and Muslim heresiological reports, Syriac texts, and Manichaean writings, he provides a detailed analysis of both the Qumran and Manichaean rescensions of the work, demonstrating additional interdependencies and suggesting new narrative arrangements. He addresses a series of quotations from an unnamed Manichaean source found in a paschal homily of the sixth-century Monophysite patriarch Severus of Antioch and a narrative from Thoeodore bar Konai. Reeves demonstrates that the motifs of Jewish Enochic literature, in particular those of the story of the Watchers and Giants, form the skeletal structure of Mani's cosmological teachings, and that Chapters 1 to 11 of Genesis fertilized Near Eastern thought, even to the borders of India and China.