DEAD-Box RNA Helicases in Bacillus subtilis Have Multiple Functions and Act Independently from Each Other

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is limited. In this study, we investigated the role of the four DEAD-box RNA helicases in the Gram-positive model organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. The deletion of cshA, cshB, or yfmL in particular leads to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis, suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype, and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis, we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.     

Endopeptidase Activities of Botulinum Neurotoxin Type B Complex,Holotoxin, and Light Chain

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most toxic substances known to humans and block the release of neurotransmitters, resulting in flaccid muscle paralysis. There are seven serotypes of BoNT, designated A to G, which are serologically distinct. An antitoxin against one serotype does not work on other serotypes. Different BoNT serotypes differ in their amino acid sequences, their substrates, or cleavage sites on the same substrate. Of the seven serotypes, BoNT type A (BoNT/A), BoNT/B, BoNT/E, and BoNT/F are known to cause human botulism (9). The extreme lethality of BoNTs makes them potent bioterror agents. BoNT/A and BoNT/B are two serotypes which have been approved by the Food and Drug Administration (FDA) for cosmetic purposes and for treatment of a wide range of neuromuscular diseases, including cervical dystonia (3).Like other BoNT serotypes, BoNT/B is secreted by the bacteria as a complex of the holotoxin and several nontoxic proteins called neurotoxin-associated proteins (NAPs). The NAPs protect the holotoxin from harsh environmental conditions, such as the high temperature, low pH, and multiple proteases present in the gastrointestinal tract (14, 17). The holotoxin, of about 150 kDa, can be obtained by removing the non-covalently bound accessory proteins with ion-exchange chromatography. The 150-kDa polypeptide chain consists of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC), which are synthesized as a single polypeptide chain but nicked by endogenous or exogenous proteases and remain linked through a disulfide bond (Fig. ​(Fig.1).1). The HC binds the receptors on neuronal cells and helps translocate the LC into the cell. The BoNT/B LC cleaves the vesicle-associated membrane protein (VAMP), also called synaptobrevin. VAMP is necessary for the docking and fusion of synaptic vesicles to plasma membrane at the neuromuscular junctions for neurotransmitter release. Once the VAMP is cleaved, the neurotransmitters in synaptic vesicles cannot be released, resulting in flaccid paralysis that can be fatal.Open in a separate windowFIG. 1.Schematic diagram of BoNT/B pure toxin. Dark gray, light chain; light gray, heavy chain; hatch-marked box, the active site of the toxin. The 50-kDa light chain and 100-kDa heavy chain are linked through a disulfide bridge as well as a covalent bond. The latter is partially nicked by bacterial proteases before the toxin is secreted.Strains producing BoNT/B can be nonproteolytic or proteolytic (4). BoNT/B from nonproteolytic strains occurs as a single polypeptide chain of 150 kDa. BoNT/B secreted by proteolytic strains is a mixture of the single polypeptide chain and a dichain in which the peptide bond linking the HC and LC has been nicked by proteases produced by the bacteria (Fig. ​(Fig.1).1). The single polypeptide chain in both nonproteolytic and proteolytic cultures can be converted to the dichain form through in vitro trypsinization. The HC and LC in the dichain can be further separated by breaking the disulfide bond with a reducing agent such as dithiothreitol (DTT) and treating it with chaotropic reagents such as urea (10).The complex, holotoxin, and LC are three different forms of BoNT/B with endopeptidase activity, although LC is the only active unit in all three forms. The complex is the native form of the toxin, which causes botulism. It is also the main component of the only licensed drug with BoNT/B currently available (2). The complex, holotoxin, and LC of BoNT/B have all been extensively used to develop methods to detect this serotype or to screen for inhibitors against the toxin (1, 5, 7, 8, 13, 15, 16). Since different forms of the toxin were used by different researchers, it is difficult to compare the sensitivities of different detection methods or the efficacies of different inhibitors. Therefore, in this study, the activities of BoNT/B complex, holotoxin, and LC were compared under the same conditions for the first time. The results suggest that the endopeptidase activity with a peptide substrate varies substantially depending on whether BoNT/B is used in its native complex form, its isolated holotoxin form, or a separated LC form. The LC form was the most active form of the endopeptidase under the conditions used.     

A Simple,Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.     

B型肉毒毒素保护性抗原Hc在大肠杆菌中的可溶性高表达

目的：通过序列优化及表达条件改进,在大肠杆菌中高效可溶性表达B型肉毒毒素保护性抗原He（Bont／B-He）。方法：对Bont／B-He基因片段优化,用大肠杆菌常用密码子替换稀有密码子,并将（C＋C）含量由76．2％降至56-3％;人工合成多条具有重叠互补序列的寡核苷酸片段,采用重叠延伸PCR方法获得了Bont／B-He的全长基因,并构建了原核可溶性表达载体;将经酶切和测序鉴定正确的重组质粒转化大肠杆菌B121（DE3）感受态细胞,用IPTC诱导Bont／B-He的表达并进行纯化及Western印迹鉴定。结果和结论：目的蛋白在大肠杆菌B121（DE3）中获得了可溶性高表达,占菌体裂解液上清总蛋白的26．7％,表达量达到30mg／L,是目前国内外已知表达的最高水平;经Ni柱一步纯化后,目的蛋白纯度可达到80.3％,为肉毒毒素中和抗体的制备及亚单位疫苗的研究奠定了基础。     

Gangliosides and Other Lipids of the Growth Cone Membrane

Growth cone membranes, derived from growth cone particles isolated from 16- to 18-day-old fetal rat brain, were found to be rich in overall lipid content with a lipid-to-protein ratio of 3.5. The phospholipid-to-cholesterol ratio indicated considerably less cholesterol than plasma membranes from mature neurons. All major classes of phospholipid were present in the usual proportions except sphingomyelin, which could not be detected. Gangliosides expressed in relation to protein were present at somewhat higher levels compared to previously reported values for synaptic plasma membranes (73 versus 44 micrograms/mg protein), but when related to phospholipid their level was well below that of the latter (26 versus 62 micrograms/mg phospholipid). The ganglioside pattern was generally similar to that of mature synaptic membranes except for the presence of relatively more GD3 and less GD1a, a phenomenon also observed in whole fetal brain of the same age. Several neutral glycosphingolipids were detected, glucosylceramide being the major one of this group. Their total level in growth cone membranes was roughly comparable to that of gangliosides, but unlike the latter their concentration in whole brain decreased with development. For comparison we analyzed the ganglioside composition of mixed membrane fractions from the same fetal brains and found no significant differences between these and growth cone membranes, suggesting that these glycoconjugates are not localized specifically in the growth cones. Neutral glycosphingolipids, on the other hand, appeared somewhat more concentrated in growth cones than in the mixed membranes.     

Bilaterally Symmetric Populations of Chicken dI1 (Commissural) Axons Cross the Floor Plate Independently of Each Other

Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1 (commissural) axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to “double” in ovo electroporate the embryonic chicken spinal cord to independently manipulate the rate of growth of the two bilateral populations of dI1 axons. Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.     

Status of Cys Residues in the Covalent Structure of Botulinum Neurotoxin Types A, B, and E

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345–2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8°C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine·HCl. In all three NT serotypes the number of Cys residues titrated with 5,5-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5–10 and 5.4–6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine·HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4–8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine·HCl in the absence of any added reducing agent. We propose that -S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine·HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.     

肉毒毒素蛋白受体syt II N端片段基因的合成及其在大肠杆菌中的融合表达

本研究旨在构建肉毒毒素蛋白受体sytII N端片段的原核表达载体,并在大肠杆菌pMAL-c2x系统中表达MBP-Syt融合蛋白。根据GenBank中已报道的人syt II基因序列,截取N端氨基酸序列,依据大肠杆菌的偏爱密码子,设计引物人工合成全基因,将全长基因克隆至原核表达载体pMAL-c2x中,重组质粒转化大肠杆菌E.coli ER2566,IPTG诱导表达。表达产物经Amylose Resin亲和层析进行纯化,SDS-PAGE和免疫印记对其进行鉴定,并对该蛋白进行活性的初步分析,为进一步研究毒素与受体相互作用的机制奠定基础。     

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

鹭鸶和鱼相安无事

在阳烂,最令我们不能忘怀的是那终日不绝于耳的鸟叫,尤其是那些成群翻飞的鹭鸶,它们发出的咔咔声总与家鸭嘶哑的叫声相和。未到阳烂之前,我们的向导兼翻译龙建云早就告诉我们,侗家的生活离不开鱼,他们一直公认左手握着糯米团,右手掂着陈年腌鱼才是人人向往的甜美生活。侗家爱吃鱼,也精通于养鱼,     

New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated with C. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models of C. botulinum neurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making.     

Characterization of the Genes Encoding the Botulinum Neurotoxin Complex in a Strain of Clostridium botulinum Producing Type B and F Neurotoxins

The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins. With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21. Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C. botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C. botulinum. These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous. By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin. It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C. botulinum type A(B), possibly indicating their common ancestry. Received: 30 March 1998 / Accepted: 21 May 1998     

Sensitive Detection of Botulinum Neurotoxin Types C and D with an Immunoaffinity Chromatographic Column Test

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml⁻¹ was achieved in culture supernatants.     

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay''s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT''s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.     

Production and Purification of the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype B, Expressed in the Methylotrophic YeastPichia pastoris

A recombinant H_cfragment of botulinum neurotoxin, serotype B (rBoNTB(H_c)), has been successfully expressed in a Mut⁺strain of the methylotrophic yeastPichia pastorisfor use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure H_cfragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified H_cfragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.     

Dogs and Owners Resemble Each Other in the Eye Region

ABSTRACT

Although several studies have shown perceived facial resemblance between photos of purebred dogs and their owners, none of these studies has clarified which part of the face is critical for such an impression to arise. The series of experiments presented here identified the critical feature underlying the perceived dog–owner facial resemblance. In total, 502 Japanese undergraduate students participated in the study. Two sets of 20 dog–owner photo pairs had been color-printed on a test sheet: the dogs varied in breed and the owners were Japanese males and females in their twenties to sixtieswho were unknown to the participants. One set comprised real pairs, while the other was a fake set (i. e., each dog was paired with another dog's owner). As in a previous study, the majority of participants (from two-thirds to three-quarters) reported that the former set was more similar than the latter. This was also the case even when the mouth regions of the owners' faces were masked by black bars. However, masking the eye regions of either dogs or owners reduced the choice performance to a chance level of around 50%. Furthermore, choice performance was equally good and above the chance level, regardless of whether the entire faces were shown or only the eye regions of the dogs and owners were visible. These results strongly suggest that dogs and owners resemble each other in the eye region. This finding also ruled out the possibility that any nonphysiognomic features (e. g., owners' hairstyles and people's stereotypical concepts about dog ownership) or similarity of obesity are determinate for the perceived dog–owner resemblance.     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Ammonia from Iron(II) Reduction of Nitrite and the Strecker Synthesis: Do Iron(II) and Cyanide Interfere with Each Other?

The question of whether the production of ammonia, from the reduction of nitrite by iron(II), is compatible with its use in the Strecker synthesis of amino acids, or whether the iron and the cyanide needed for the Strecker synthesis interfere with each other, is addressed. Results show that the presence of iron(II) appears to have little, or no, effect on the Strecker synthesis. The presence of cyanide does interfere with reduction of nitrite, but the reduction proceeds at cyanide/iron ratios of less than 4:1. At ratios of about 2:1 and less there is only a small effect. The reduction of nitrite and the Strecker can be combined to proceed in each other's presence, to yield glycine from a mixture of nitrite, Fe⁺², formaldehyde, and cyanide.