A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60°C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.

Two pairs of DuPont Zorbax PSM Bimodal gel permention chromatography columns, packed with porous silica particles have been used fo the fractionation of dextran. High initial efficiencies decreased with use and shrinkage of the bed suggested silica was dissolving. The eluent required a certain ionic strength to prevent some of the dextran being excluded.

A number of rigid column packings (Spherosil-Porasil and DuPont SEC) and semi-rigid packings (two types of Spheron gel and Hydrogel) have been used for the molecular-weight characterisation of dextran by aqueous gel permeation chromatography. Our experience with these packings is reported and the various problems encountered, and their possible causes, are discussed. Our criteria for an acceptable packing have been fractionating range, efficiency, short analysis time and, of particular importance, long-term stability. None of the column packings considered was found to be ideal.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA New Process for the Continuous Fractionation of DextranPhilip E. Barker, F. John Ellison, and Brian W. HattCite this: Ind. Eng. Chem. Process Des. Dev. 1978, 17, 3, 302–309Publication Date (Print):July 1, 1978Publication History Published online1 May 2002Published inissue 1 July 1978https://pubs.acs.org/doi/10.1021/i260067a015https://doi.org/10.1021/i260067a015research-articleACS PublicationsRequest reuse permissionsArticle Views65Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options Get e-Alerts

The construction and operation of a liquid preparativescale continuous chromatograph is described. The chromatographic bed is arranged in a series of interconnected vertical columns and is rotated whilst one end of the tube bundle is closed by a stationary plate through which liquids enter and leave the bed. The problem of maintaining an efficient seal is described. The chromatograph was commissioned by continuously fractionating dextran on porous silica beads with water as eluent. Results show that as the feed concentration was increased, the fractionation obtained deviated markedly from that expected from basic GPC theory, but that the relative molecular mass distribution of the feed was still narrowed with feed concentrations of 20% w/v.

Equipment which has been used or suggested for preparative scale chromatography is reviewed. Particular attention is paid to one form of equipment for continuous counter-current chromatography which employs a moving mechanical seal. The theoretical effects of practical operating conditions and the mode of operation of this equipment are discussed. The influence of these effects on polymer fractionation is also considered. A theoretical model, based on the equilibrium stage concept, is proposed for the simulation of continuous gel permeation chromatography of a binary feed on such an apparatus. Examples of the results obtained from the computer simulation are given.

The interaction of benzeneboronic acid, 4-methoxybenzeneboronic acid, and 3-nitrobenzeneboronic acid with d-glucose, d-mannose, and d-fructose at various pH values has been investigated by means of optical rotation methods. The effects of (a) various molar ratios of sugar and acid and (b) overall concentration on the extent of complex formation are reported.

Benzeneboronic acid, 4-methoxybenzeneboronic acid, 3-nitrobenzeneboronic acid, and sulphonated benzeneboronic acid have been used to displace the pseudo-equilibria established in aqueous alkali between d-glucose, d-fructose, and d-mannose to give greatly increased yields of d-fructose. The effect of reaction temperature, pH, overall concentration, and molar ratio of acid:sugar on the yield of d-fructose has been investigated by using an automated assay for d-fructose.