John H. Hash

The repeating sequence of the capsular polysaccharide of Staphylococcus aureus M

The anomeric configuration of the sugar residues of the capsular polysaccharide antigen of Staphylococcus aureus M were established by 13C-n.m.r. spectroscopy, and the linkage positions by g.l.c.-m.s. after methylation, indicating a leads to 4)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyluronic acid)-(1 leads to 4)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyluronic acid)-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-fucopyranosyl)-(1 leads to repeating unit. A taurine residue is linked by an amide bond, on the average, to every fourth 2-acetamido-2-deoxy-D-galactopyranosyluronic acid residue.

A review of the molecular structure of tetanus toxin

SummaryA discontinuous preparative polyacrylamide gel electrophoresis system has been developed and used to purify both the nicked and unnicked forms of tetanus toxin. The system was also used to prepare purified H and L chain peptides from the nicked toxin. The results show that the endogenous protease(s), which convert unnicked toxin to the nicked form, produce multiple species of nicked toxin, and heterogeneity in the H and L chains. The major amino termini of the toxins and their peptide components are: extract toxin, proline; filtrate toxin, proline, serine and asparagine; L chain, proline; and H chain, serine and asparagine. The L chain is located in the amino terminal position of the toxin molecule and the H chain the carboxy terminal end. A model is proposed to explain these results. Using the analytical ultracentrifuge, we have determined the molecular weights of extract and filtrate toxins to be 140 000 ± 5 000 and 128 000 ± 3 000, respectively. Using S DS-polyacrylamide gel electrophoresis we estimate the molecular weights of the H and L chains to be 87 000 and 48 000 daltons, respectively.Circular dichroic spectra of the toxins and their peptide components indicate that: the major tryptophanyl band in the toxin is contributed almost entirely by the H chain, the microenvironments of all the aromatics and disulfides in the two toxins appear to have small if any differences, the two toxins show little difference in their ordered secondary structure, and the two peptides when separated from one another still retain 80% of the helical structure that is present in the intact toxin but show a considerable loss of β-structure.The crystalline form of the nicked toxin has a hexagonal symmetry with two dimensional reciprocal lattice constants of 1/150 Å−1 and 1/150 Å−1. The crystals appear to belong to the two dimensional plane group P6 suggesting that each unit cell contains 6 or a multiple of 6 toxin molecules.

Use of a ternary gradient for the separation of phenylthiohydantoin-amino acids, including the methyl esters of aspartic and glutamic acids, by high-performance liquid chromatography

Abstract The phenylthiohydantoin (PTH) derivatives of the common amino acids can be resolved in a single high-performance liquid chromatographic analysis by elution from a cyanopropylsilane column with a ternary gradient of methanol, acetonitrile, and ammonium acetate. The system is compatible with an automated sequencer-automated converter combination that produces the methyl esters of PTH-aspartic and PTH-glutamic acids.

Characterization of tetanus toxins and toxin components by amino terminal analyses

Abstract Extract tetanus toxin, filtrate tetanus toxin, and the heavy and light chains of filtrate toxin were analyzed for their amino termini with 4- N,N -dimethylaminoazobenzene-4′isothiocyanate and phenylisothiocyanate. Extract toxin (intracellular toxin) is a single-chain polypeptide with proline as the amino terminus. Filtrate toxin (extracellular toxin) is a mixture of species produced by endogenous proteases, and showed three major amino terminal residues, proline, asparagine, and serine. Cleavage points in the filtrate toxin molecule appear to be on either side of a disulfide bond. Reductive and nonreductive preparative electrophoresis of filtrate toxin produce different species of light and heavy chains. The light chains have a single amino terminus of proline, indicating that the light chain is the amino terminal portion of the toxin molecule. The heavy chains showed no proline but rather asparagine and serine as the major amino termini. Small amounts of other amino terminal residues were present, indicating microheterogenity at the cleavage sites in the toxin. The results permit the construction of a model of tetanus toxin which is consistent with the fragments obtained from either reductive or nonreductive preparative electrophoresis of filtrate toxin.

The use of an amino acid analyzer for the rapid identification and quantitative determination of chitosan ollgosaccharides

Abstract The oligosaccharides of chitosan, glucosamine through chitohexaose, were separated and quantitated with the use of an amino acid analyzer. The oligosaccharides can be satisfactorilly resolved with a step gradient using two buffers, but resolution is improved with a linear buffer gradient. Using experimentally determined color factors, the method permits the rapid quantitation and identification of small amounts of chitosan oligosaccharides.

Purification of tetanus toxin and its peptide components by preparative polyacrylamide gel electrophoresis

Abstract A discontinuous preparative gel electrophoresis system has been devised and used successfully to separate the different tetanus toxin forms and fragments into highly purified preparations. A major feature of the system is the interaction of toxin, a suitable reducing agent and a critical concentration of denaturant during electrophoresis. With this procedure, filtrate (nicked) toxin has been separated into two distinct, but closely related molecular species. They appear to be nicked close to but on either side of an interchain disulfide bond, yielding heavy and light chains. The heavy- and light-chain components of each form of nicked toxin have been prepared and characterized. The system was also used to prepare extract (unnicked) toxin to a degree of purity not previously achieved in this laboratory. Nicked and unnicked toxin as well as the two forms of both heavy and light chain can consistently be prepared in sufficient purity and quantity to allow extensive biological, chemical, and physical characterization of each.

Conformation of diphtheria toxin and an enzymically-active fragment

Diphtheria toxin is synthesized by strains of C. diphtheria which are lysogenic for bacteriophage carrying the tax gene [l-3] . The toxin, which contains two disulfide bonds, is released into the extracellular medium as a single-chain polypeptide with mol. wt -60 000 [4,5] . In this form, toxin is inactive [6,7] but can be activated by mild proteolysis and reduction [S] . This yields two fragments, designated A and B, with respective mol. wt -21 000 and -38 000 [l-7] . Fragment A, which is derived from the amino terminus of toxin and has been sequenced [9], catalyzes the covalent linkage of the adenosine diphosphate ribose moiety of NAD+ to elongation factor EF-2 [ 10,l l] . This inactivates EF-2 and thus terminates protein biosynthesis in cell-free extracts of eukaryotes [l-3] . The present study is aimed at elucidating certain conformational aspects of diphtheria toxin and fragment A, both of which are soluble in aqueous solution; in contrast, fragment B is relatively insoluble [2,3]. Circular dichroic (CD) spectra have been obtained for both toxin and fragment A in the near (250-3 10 nm) and far (200-250 nm) ultraviolet (uv), where the ellipticity is strongly influenced by tertiary and secondary structure, respectively. Analysis of the toxin CD spectrum gave 20% cu-helicity and 25-3%@tructure; fragment A exhibited diminished ol-helicity and a somewhat elevated content of /?-

Enzymatic fragmentation of tetanus toxin. Identification and characterization of an atoxic, immunogenic fragment.

SummaryPurified filtrate tetanus toxin was subjected to limited digestion with papain and the resulting fragments were separated by gel exclusion chromatography and characterized. One atoxic fragment was shown to react with antiserum against tetanus toxoid and was capable of inducing antibodies in rabbits that neutralized native tetanus toxin. The fragment had an estimated molecular weight of 56,000 by SDS polyacrylamide gel electrophoresis and 62,000 by sedimentation equilibrium. In the presence of a reducing agent, the fragment yielded two components with approximatec molecular weights of 23,000 and 32,000. Thus, it appears that the atoxic, immunogenic fragment is composed of two peptides joined by at least one disulfide bond. The fragment was examined by circular dichroism and data analysis indicated the presence of considerable β-structure, but little, if any, α-helicity. This is significantly different from the estimates for filtrate toxin, 29% α-helicity and 23% β-structure. Above 250 nm, the circular dichroic spectrum of the fragment was also distinct from that of intact toxin.

Chapter V Liquid Scintillation Counting in Microbiology

Publisher Summary This chapter discusses the liquid scintillation counting in microbiology. Liquid scintillation counting is a method of detecting radioactivity with a solution of certain chemicals and a photomultiplier tube. A scintillator is a material that emits a brief pulse of fluorescent light, or scintillation, when it interacts with a high-energy particle or quantum. The liquid scintillator may be simple or relatively complex, but it must have two essential ingredients: solvent and scintillation solute. In external liquid scintillation counting, the source of radiation is external to the liquid scintillator. These are usually large volume systems. Internal liquid scintillation counting applies to the situation where the source of radiation is intimately mixed with the liquid scintillator. These are usually small volume systems. The intrinsic advantage of internal liquid scintillation counting over other methods of detection is that it puts the source of radiation in intimate contact with the detector. The single most important characteristic that a scintillation solvent must possess is the ability to accept energy from incident ionizing radiation and transfer it to a dissolved scintillator. In addition, other characteristics, such as proper solvent properties in relation to the sample or a small absorption coefficient for the light the solute emits may be superimposed upon this basic requirement.