Article

A Protein Sequenator

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Abstract

The protein sequenator is an instrument for the automatic determination of amino acid sequences in proteins and peptides. It operates on the principle of the phenylisothiocyanate degradation scheme. The automated process embraces the formation of the phenylthiocarbamyl derivative of the protein and the splitting off of the N-terminal amino acid as thiazolinone. The degradation proceeds at a rate of 15.4 cycles in 24 hours and with a yield in the individual cycle in excess of 98%. The material requirements are approximately 0.25 μmoles of protein. The thiazolinones are converted to the corresponding phenylthiohydantoins in a separate operation, and the latter identified by thin layer chromatography. The process has been applied to the whole molecule of apomyoglobin from the humpback whale, and it has been possible to establish the sequence of the first 60 amino acids from the N-terminal end.

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... 1967年就已经有液体旋转杯法, 实现了自动蛋白测 序 [19] (图1 [20] . 另外, 对于蛋白翻译 后修饰(protein translational modifications, PTMs)也较 难准确分析 [21,22] . ...
... 目前质谱技术是用以测定蛋白质一级结构的主流 方法, 主要有两种思路: 自下而上蛋白质组学分析方法 (bottom-up proteomics)和自上而下蛋白质组学分析方 法(top-down proteomics) [27,28] (图2). 前者是目前质谱测 序的主流技术, 其思路着眼于通过消化和酶解等手段, 将蛋白质的一级结构打散为小的多肽片段, 再进行液 图 1 旋转杯法蛋白质自动测序系统示意图 [19] Figure 1 Diagram of the automatic protein sequenator using a spinning cup [19] 相色谱分离和在线二级质谱分析. 所获多肽链的序列 信息可以通过搜索引擎(例如Sequest和Mascot)与已知 蛋白质的序列进行比对, 进而实现蛋白质序列的鉴 定 [29] . ...
... 目前质谱技术是用以测定蛋白质一级结构的主流 方法, 主要有两种思路: 自下而上蛋白质组学分析方法 (bottom-up proteomics)和自上而下蛋白质组学分析方 法(top-down proteomics) [27,28] (图2). 前者是目前质谱测 序的主流技术, 其思路着眼于通过消化和酶解等手段, 将蛋白质的一级结构打散为小的多肽片段, 再进行液 图 1 旋转杯法蛋白质自动测序系统示意图 [19] Figure 1 Diagram of the automatic protein sequenator using a spinning cup [19] 相色谱分离和在线二级质谱分析. 所获多肽链的序列 信息可以通过搜索引擎(例如Sequest和Mascot)与已知 蛋白质的序列进行比对, 进而实现蛋白质序列的鉴 定 [29] . ...
... Amino acid sequence analyses extended for 6 to 39 positions (a few included the first hypervariable region) depending upon the quantity of light chain obtained from the focused fractions. Sequences obtained from light chains isolated from serum prior to immunization immunoglobulins or from specifically purified unfractionated antibodies were heterogeneous (2 to 4 residues at most positions), while those from the major focused antibody fractions were as homogeneous, for most of the first 25 to 30 Preparative liquid isoelectric focusing has been used to prepare a number of rabbit antihapten antibody fractions which display properties suggestive of structural, charge, and functional homogeneity (l-3). ...
... Determination of Amino-terminal Light Chain Sequences--Light chains (0.14 to 0.33 pmole) were sequenced on a Beckman model 890 protein sequencer (30). All reagents and solvents were purchased from Beckman Instruments. ...
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... N-terminal sequencing was performed according to Edman's degradation method [45], using an automatic protein sequenator (PPSQ-33A, Shimadzu, Kyoto, Japan), following the manufacturer's instructions. The obtained sequence was compared to databases through the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi) ...
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... The HPLC purified fraction was loaded onto a ProSorb device (Applied Biosystems, Foster City, CA). The ProSorb membrane was then loaded onto a protein sequencing instrument, Shimadzu PPSQ-53A (Shimadzu, Columbia, MD) to carry out an Edman degradation sequencing method (Edman and Begg, 1967). The N-terminal amino acids were determined by comparing the retention times of the resulting PTH-amino acid from each Edman degradation cycle with the retention times of a standard mixture of 19 PTH-amino acids in a reference chromatogram. ...
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We offer a path-centric theory of emerging technology and organizing that addresses a basic question: when does emerging technology lead to transformative change? A path-centric perspective on technology focuses on the patterns of actions afforded by technology-in-use. We identify performing and patterning as self-reinforcing mechanisms that shape patterns of action in the domain of emerging technology and organizing. We use a dynamic simulation to show that performing and patterning can lead to a wide range of trajectories, from lock-in to transformation, depending on how emerging technology-in-use influences the pattern of action. When emerging technologies afford new actions that can be flexibly recombined to generate new paths, decisive transformative effects are more likely. By themselves, new affordances are not likely to generate transformation. We illustrate this theory with examples from the practice of pharmaceutical drug discovery. The path-centric perspective offers a new way to think about generativity and the role of affordances in organizing.
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A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.
Article
Vascular endothelial growth factors (VEGFs) are crucial molecules involved in the modulation of angiogenesis. Snake venom-derived VEGFs (svVEGFs) are known to contribute significantly to the envenoming due to their capacity of increasing vascular permeability. In our work, we isolated and analyzed the biochemical and functional properties of the VEGF from Crotalus durissus collilineatus venom (CdcVEGF). The venom was fractionated by reversed phase chromatography on FPLC system (Fast Protein Liquid Chromatography) and the eluted fractions were submitted to an ELISA assay using an anti-VEGF-F antibody, for identification of svVEGF. Positive fractions for svVEGF were submitted to SDS-PAGE and to an anion exchange chromatography to isolate the molecule. The subfractions were analyzed by ELISA and SDS-PAGE and six of them presented svVEGFs, named CdcVEGF1 (Q23-3), CdcVEGF2 (Q24-3), CdcVEGF3 (Q24-4), CdcVEGF4 (Q25-3), CdcVEGF5 (Q25-4), and CdcVEGF6 (Q25-5). Their structural characterization was accomplished by mass spectrometry analysis using MALDI-TOF to determine their molecular masses and UPLC-ESI-QTOF to determine their amino acid sequence. Interestingly, all isolated CdcVEGFs induced angiogenesis on HUVEC cells through tube formation on Matrigel when compared to culture medium (negative control). Moreover, CdcVEGF2 and CdcVEGF3 also induced a significant increase in tube formation when compared to the positive control (basic fibroblast growth factor - bFGF). Additionally, crotalid antivenom produced by the Instituto Butantan was able to recognize CdcVEGFs, demonstrating to be immunogenic. This study demonstrates that snake venom cocktail can reveal novel and important molecules, which are potential molecular tools to study diverse biological processes, such as angiogenesis.
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The profound effects of and distress caused by the global COVID-19 pandemic highlighted what has been known in the health sciences a long time ago: that bacteria, fungi, viruses, and parasites continue to present a major threat to human health. Infectious diseases remain the leading cause of death worldwide, with antibiotic resistance increasing exponentially due to a lack of new treatments. In addition to this, many pathogens share the common trait of having the ability to modulate, and escape from, the host immune response. The challenge in medical microbiology is to develop and apply new experimental approaches that allow for the identification of both the microbe and its drug susceptibility profile in a time-sensitive manner, as well as to elucidate their molecular mechanisms of survival and immunomodulation. Over the last three decades, proteomics has contributed to a better understanding of the underlying molecular mechanisms responsible for microbial drug resistance and pathogenicity. Proteomics has gained new momentum as a result of recent advances in mass spectrometry. Indeed, mass spectrometry-based biomedical research has been made possible thanks to technological advances in instrumentation capability and the continuous improvement of sample processing and workflows. For example, high-throughput applications such as SWATH or Trapped ion mobility enable the identification of thousands of proteins in a matter of minutes. This type of rapid, in-depth analysis, combined with other advanced, supportive applications such as data processing and artificial intelligence, presents a unique opportunity to translate knowledge-based findings into measurable impacts like new antimicrobial biomarkers and drug targets. In relation to the Research Topic “Proteomic Approaches to Unravel Mechanisms of Resistance and Immune Evasion of Bacterial Pathogens,” this review specifically seeks to highlight the synergies between the powerful fields of modern proteomics and microbiology, as well as bridging translational opportunities from biomedical research to clinical practice.
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Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The Iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAT (NEAr-iron Transporter) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints, and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route towards IsdH do not play a critical role in the rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.
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Omics is among the rapidly evolving field of study routinely tested and employed by the global scientific community to understand biological mysteries. Mass spectrometry (MS) is one of the key technologies that has enabled scientists to investigate and delineate critical aspects of biology through proteomics, metabolomics, or lipidomics approaches. Molecular and cellular heterogeneity within an organism or cell contributes to biological consequences such as growth, development, diseases, disorders, mutations, stress, etc. Recent advancements in mass spectrometry have accelerated the comprehensive understanding of such heterogeneities and consequences. In this chapter, we will focus on such developments and advances in mass spectrometry that are enabling scientists to attain thorough visibility of biology through MS-based omics approaches.
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Bereits 1940 war man sich einig, dass Proteine aus Aminosäuren bestehen und dass die Aminosäuren über die sog. Peptidbindung verknüpft sind (◘ Abb. 15.1). Man wusste, dass die so vorhandenen kettenartigen Moleküle an einem Ende, das als N-terminales Ende bezeichnet wird, eine freie Aminogruppe tragen, und an dem anderen, dem C-terminalen Ende, eine freie Carboxygruppe. Keineswegs einig war man sich zu dieser Zeit hingegen, ob ein bestimmtes Protein aus einem Gemisch verschiedener Polymere besteht, die zwar eine definierte Anzahl und Art von Aminosäuren beinhalten, deren Reihenfolge aber ganz unterschiedlich sein kann, oder aus einer einzigen Spezies von Molekülen, die eine ganz definierte Aminosäuresequenz aufweisen.
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By many parameters, chemistry is a central science that interfaces several disciplines with overlapping borders to a different extension to each one of them. On the other hand, systems biology became a new concept to a holistic approach to understanding life. One of the overlapping borders between chemistry and biology is bioanalytical chemistry that could be considered the analytical tools and methods to provide the data necessary in systems biology. In this chapter, we will explore further this concept of analytical chemistry for life sciences to the point that we could consider as bioanalytical systems! We will also make a brief introduction to the critical elements of this exciting field, such as the omics sciences and high-resolution, high-throughput instrumentation, keeping in mind that other chapters in this book will go deeper into the analytical techniques and methods.
Article
How many different proteins can be produced from a single spliced transcript? Genome annotation projects overlook the coding potential of reading frames other than that of the reference open reading frames (refORFs). Recently, alternative open reading frames (altORFs) and their translational products, alternative proteins (altProts), have been shown to carry out important functions in various organisms. AltORFs overlapping refORFs or other altORFs in a different reading frame may be involved in one fundamental mechanism so far overlooked. A few years ago, it was proposed that altORFs may act as building blocks for chimeric (mosaic) polypeptides, which are produced via multiple ribosomal frameshifting events from a single mature transcript. We adopt terminology from that earlier discussion and call this mechanism mosaic translation. This way of extracting and combining genetic information may significantly increase proteome diversity. Thus, we hypothesize that this mechanism may have contributed to the flexibility and adaptability of organisms to a variety of environmental conditions. Specialized ribosomes acting as sensors probably played a central role in this process. Importantly, mosaic translation may be the main source of protein diversity in genomes that lack alternative splicing. The idea of mosaic translation is a testable hypothesis, although its direct demonstration is challenging. Should mosaic translation occur, we would currently highly underestimate the complexity of translation mechanisms and thus the proteome.
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The determination of the first protein sequence (insulin) was a landmark achievement that confirmed proteins were composed of a specific order of amino acid residues and lead to the development of methods, many eventually automated, for the rapid sequence analysis of a wide variety of proteins. Much of this technology was devoted to the isolation of proteins, and the peptides derived from them, that have remained indispensable tools even today. In the late ‘70s, methodology for the determination of nucleic acid sequences was perfected, which largely supplanted the chemical approaches and lead to an enormous expansion of the known protein sequences, albeit they were inferred rather than directly determined. Subsequently, advances in mass spectrometry made direct analyses of peptides and proteins possible allowing for high throughput unbiased identification of proteins (from peptide sequences) in complex mixtures and for the identification and localization of post-translational modifications. Refinements of this technology are now being applied to single cells and to the determination of low abundance proteins, even single molecules, within them. Sequence data, starting from the earliest experiments, has provided ever greater insight into evolutionary processes.
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Three-dimensional protein structure prediction from amino acid sequence has been a thought-provoking task for decades, but it of pivotal importance as it provides a better understanding of its function. In recent years, the methods for prediction of protein structures have advanced considerably. Computational techniques and increase in protein sequence and structure databases have influence the laborious protein structure determination process. Still there is no single method which can predict all the protein structures. In this review, we describe the four stages of protein structure determination. We have also explored the currenttechniques used to uncover the protein structure and highpoint best suitable method for a given protein.
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Proteases regulate other proteases directly via cleavage or indirectly via cleavage and the inactivation of protease inhibitors. These proteolytic interactions enable functional cross-talk between proteases and have been well established in proteolytic cascades. In addition, far-reaching interactions beyond classically defined protease cascades have been identified, suggesting a larger network of proteases—the protease web. Protease cross-talk in this network has potentially detrimental effects on protease biology in health and disease. This chapter outlines the evidence for the protease web, its effect on drug discovery and protease substrate identification, and computational approaches for modeling and predicting proteolytic interactions within this network.
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This chapter introduces the principles and practice of various analytical methods important for characterizing herbal biomolecules that include both primary and secondary metabolites. It offers scientists a survey of common analytical techniques including ultraviolet-visible, fluorescence, polarimetry, circular dichroism, fourier transform-infrared spectroscopy, nuclear magneteic resonance, single-crystal X-ray diffraction, mass spectrometry, liquid chromatography, gas chromatography, thin layer chromatography, immunoassays, gel and capillary electrophoresis and DNA and protein sequencing. The discussion of each technique is further illustrated by its application to characterize specific herbal biomolecules, including Amaryllidaceae alkaloids, tropane alkaloids, terpenes, phenolic compounds, glycosides, polysaccharides, DNA, RNA, protein and peptides. The chapter highlights the critical roles of analytical techniques in characterizing the complex structures and determining the composition of the herbal biomolecules to better understand herbal quality and biological action in healthcare and diseases.
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Antimicrobial peptides (AMPs) are widespread in multicellular organisms. These structurally diverse molecules are produced as the first line of defense against pathogens such as bacteria, viruses, fungi, and parasites. Also known as host defense peptides in higher eukaryotic organisms, AMPs display immunomodulatory and anticancer activities. During the last 30 years, technological advances have boosted the research on antimicrobial peptides, which have also attracted great interest as an alternative to tackling the antimicrobial resistance scenario mainly provoked by some bacterial and fungal pathogens. However, the introduction of natural AMPs in clinical trials faces challenges such as proteolytic digestion, short half-lives, and cytotoxicity upon systemic and oral application. Therefore, some strategies have been implemented to improve the properties of AMPs aiming to be used as effective therapeutic agents. In the present review, we summarize the discovery path of AMPs, focusing on preclinical development, recent advances in chemical optimization and peptide delivery systems, and their introduction into the market.
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Motivation The evolutionary processes of mutation and recombination, upon which selection operates, are fundamental to understand the observed molecular diversity. Unlike nucleotide sequences, the estimation of the recombination rate in protein sequences has been little explored, neither implemented in evolutionary frameworks, despite protein sequencing methods are largely used. Results In order to accommodate this need, here I present a computational framework, called ProteinEvolverABC, to jointly estimate recombination and substitution rates from alignments of protein sequences. The framework implements the approximate Bayesian computation approach, with and without regression adjustments, and includes a variety of substitution models of protein evolution, demographics and longitudinal sampling. It also implements several nuisance parameters such as heterogeneous amino acid frequencies and rate of change among sites and, proportion of invariable sites. The framework produces accurate coestimation of recombination and substitution rates under diverse evolutionary scenarios. As illustrative examples of usage, I applied it to several viral protein families, including coronaviruses, showing heterogeneous substitution and recombination rates. Availability ProteinEvolverABC is freely available from https://github.com/miguelarenas/proteinevolverabc, includes a graphical user interface for helping the specification of the input settings, extensive documentation and ready-to-use examples. Conveniently, the simulations can run in parallel on multicore machines. Supplementary information Supplementary information is available at Bioinformatics online.
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The acceptance in 2012 by the World Anti‐Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on P‐III‐NP and IGF‐I was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti‐doping standards, where the strict liability principle applies, is discussed. An alternative WADA accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half‐life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When an LC‐MS method became a reality for the measurement of IGF‐I, more consistency of results was assured. Measurement of P‐III‐NP is still dependant on immunoassays although work is underway to develop an LC‐MS method. The promised long‐term detection time for the biomarker assay does not appear to have been realised in practice and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P‐III‐NP and IGF‐I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the GH‐score would mandate an investigation and possible action for hGH administration.
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Mass spectrometry-based proteomics empowers deep profiling of proteome and protein posttranslational modifications (PTMs) in Alzheimer’s disease (AD). Here we review the advances and limitations in historic and recent AD proteomic research. Complementary to genetic mapping, proteomic studies not only validate canonical amyloid and tau pathways, but also uncover novel components in broad protein networks, such as RNA splicing, development, immunity, membrane transport, lipid metabolism, synaptic function, and mitochondrial activity. Meta-analysis of seven deep datasets reveals 2,698 differentially expressed (DE) proteins in the landscape of AD brain proteome ( n = 12,017 proteins/genes), covering 35 reported AD genes and risk loci. The DE proteins contain cellular markers enriched in neurons, microglia, astrocytes, oligodendrocytes, and epithelial cells, supporting the involvement of diverse cell types in AD pathology. We discuss the hypothesized protective or detrimental roles of selected DE proteins, emphasizing top proteins in “amyloidome” (all biomolecules in amyloid plaques) and disease progression. Comprehensive PTM analysis represents another layer of molecular events in AD. In particular, tau PTMs are correlated with disease stages and indicate the heterogeneity of individual AD patients. Moreover, the unprecedented proteomic coverage of biofluids, such as cerebrospinal fluid and serum, procures novel putative AD biomarkers through meta-analysis. Thus, proteomics-driven systems biology presents a new frontier to link genotype, proteotype, and phenotype, accelerating the development of improved AD models and treatment strategies.
Article
The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.
Article
Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.
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The venom of the black mamba (Dendroaspis polylepis) was fractionated by chromatography on Amberlite CG-50. Two of the toxins were purified, and their amino acid sequences were determined. Toxin α is a 60-residue protein cross-linked by four disulfide bridges; it is novel among snake venom toxins in containing 4 tyrosyl residues as opposed to the usual single tyrosyl residue. Toxin γ is a 72-residue protein with five disulfide bridges and is the first snake venom toxin isolated to contain 2 tryptophanyl residues. Toxins α and γ have subcutaneous LD50 values of 0.09 and 0.12 µg per g of mouse, respectively.
Article
In an attempt to extend the application of the phenylisothiocyanate degradation of peptides it was found necessary to study the kinetics of the conversion of phenylthiocarbamyl amino acids into phenylthiohydantoins. The conversion was found to obey first-order kinetics and to be catalyzed by hydrogen ions. A set of conditions with regard to time, hydrogen ion concentration and temperature was found, which allowed the quantitative or near quantitative conversion of all phenylthiocarbamyl amino acids into phenylthiohydantoins with the only exception of the phenylthiohydantoin of serine, which was returned in a yield of 20%.
Article
Myoglobin of Physeter catodon (sperm whale) forms monoclinic crystals with space group P2$_{1}$ (type A) in ammonium sulphate, and orthorhombic crystals with space group P2$_{1}$2$_{1}$2$_{1}$ (type B) in phosphate. Almost identical crystal forms are obtained from related species of whale. Two-dimensional Patterson projections have been computed from some principal zones of reflexions in each type of crystal. They are used to derive the chain directions of the polypeptide chains in the molecules relative to the crystal axes, and to suggest plausible methods of packing in the two forms. The data are best fitted by a molecule having dimensions about 25 $\times $ 34 $\times $ 42 angstrom, the latter dimension being parallel to the chain direction. Measurements of electron-spin resonance (Bennett & Ingram 1956) and of optical dichroism have been used to determine the orientation of the planes of the haem group and the angle between them and the chains. It is concluded that the angle is 40 to 50 degrees in the two forms, in good agreement with the value found for type F crystals in an earlier paper, namely, 41 degrees. Thus in all three crystal types A, B and F the data are consistent with the molecule having the same set of dimensions, associated with a common chain direction and a common relation between chain direction and haem group.
Article
1.1. Four different fibrinopeptides, A, AP, Y and B, have been isolated from human fibrinogen by means of chromatography and precipitation procedures.2.2. The amino acid sequence has been determined with the phenylisothiocyanate degradation method and by means of fragmentation with proteolytic enzymes and partial acid hydrolysis. The A-, AP- and Y-peptides are similar in structure, the AP-peptide being an A-peptide phosphorylated at the serine residue in Position 3 from the N-terminal end and the Y-peptide being one amino acid residue shorter than the A-peptide from the N-terminal end. The B-peptide has been found to have pyroglutamic acid as N-terminal residue.3.3. The phosphorus in human fibrinogen is partly bound to an AP-peptide and partly to other structures of the fibrinogen molecule.4.4. On basis of the results the structure of human fibrinogen and the specificity of thrombin is discussed.
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