Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.

SummaryOur study investigated the immunomodulatory activities of human plasmaderived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro-or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide ( We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities.

Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.

HAV and B19V are effectively removed by antibody-dependent 35N nanofiltration, already at intermediate antibody concentrations well below those comparable to human plasma pools for fractionation.

The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability.

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable.Sedimentation equilibrium, gel permeation chromatography, and NaDodSO,/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000k 1500 for both native and denatured enzyme.The unfolding transition at varying guanidine . HCl concentrations is characterized by a two-step profile: at 0.4 -0.8 M, partial unfolding is parallelled by inactivation; at 2.0 -2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4 -1.8 M guanidine . HC1, partial unfolding is superimposed by aggregation.The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine . HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33 %.Denaturation in 6 M guanidine . HCI, as monitored by the decrease of protein fluorescence, is fast ( < 8s). Upon reactivation after short denaturation, about 25 % of the activity is recovered in a fast initial phase (< 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by z = 29 f 3 min (20 "C). This reaction must be a relatively late event on the folding pathway, Octopine dehydrogenase is a monomeric NAD-dependent enzyme, which catalyzes the reductive condensation of pyruvate and L-arginine to D-octopine. The enzyme has been reported to occur in molluscs, nemerteans, sipunculids, and coelenterates. The biological and physiological role of octopine dehydrogenase in various organisms has been studied in detail (for a review, see [l]). Tissue-specific isoenzymes were detected in certain cephalopodes [2,3]. The enzymatic mechanism has been analyzed for octopine dehydrogenase from muscle of the bivalve Pecten maximus [4 -151. The enzyme occurs in two molecular forms which differ in their electrophoretic mobility but not in their structural and kinetic parameters [ 161. Electrophoretically different forms of octopine dehydrogenase were also identified in Pecten ,jacobaeus and some other bivalves [2,17,18].Physicochemical data have only been determined for the enzyme from P. maximus [5,16,19]. The present work summarizes physicochemical and enzymatic data for the two electrophoretically different forms of octopine dehydrogenase from the adductor muscle of P. jacoba...

Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans.

The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN. At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers. These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction. Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation. The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation. This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer. The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers. The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor. Two successive processes can be observed during reassociation of the protein upon KSCN dilution. First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS)