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Jeanne Brygier Michel Gelbcke Claude Guermant Michelle Nijs Danielle Baeyens-Volant Yvan Looze 《Applied biochemistry and biotechnology》1993,42(2-3):127-135
Carbonylimidazol-l-yl-mPEG is obtained quantitatively by reacting methoxypoly(ethyleneglycol) (mPEG) with 1.1 Eq of N,N′-carbonyldiimidazole
in the presence of a stoechiometric amount of 4-dimethyl-aminopyridine used as hypernucleophilic acylation catalyst. Carbonylimidazol-l-yl-mPEG is quite stable in aqueous solutions with half-lives up to 70 h in pHs ranging from 6.0 to 7.0 at 25‡C. From reactivity
studies toward amines with various nucleophilic strengths, it is suggested that carbonylimidazol-l-yl-mPEG may be best used to modify α-amino terminal function of proteins selectively or to introduce amino function on PEG chains. 相似文献
2.
Mohamed Azarkan Michelle Nijs Nicole Smolders Jlaude Guermant Jean Vlncentelli Yvan Looze 《Applied biochemistry and biotechnology》1996,60(2):167-183
The four cysteine proteinases, papain, chymopapain, caricain, and endoproteinase Gly-C were isolated and purified as the catalytically competent species from the commercially available latex of the tropical treeCarica papaya L. Their free thiol function (cysteine-25), which is essential for activity, was protected in the form of a mixed disulfide containing a 5 kDa polyethylene glycol (PEG) chain. The second (nonessential) free thiol function (cysteine-117) of chymopapain was blocked similarly. Caricain was also derivatized through acylation of its amino functions by PEG chains (average: 15 moles of PEG per mole of enzyme). The Chromatographic behavior of these conjugates was examined on ion-exchange and hydrophobic gels and compared to the Chromatographic behavior of the unpegylated proteinases. The results indicated that charge-shielding effects by PEG chain(s), surrounding the different proteinases, plays(play) a key role in the course of separation of pegylated and unpegylated species by ion-exchange chromatography. Similarly, PEG chain(s) is(are) able to mask hydrophobic regions on the surface of the proteinases. However, the affinity showed by PEG itself for the hydrophobic ligands immobilized on the matrix is the preponderant factor determining the behavior of the PEG-proteinases conjugates on Fractogel TSKButyl-650. 相似文献
3.
Tony Musu Mohamed Azarkan Jeanne Brygier Claudine Paul Jean Vincentelli Danielle Baeyens-Volant Claude Guermant Michelle Nijs Yvan Looze 《Applied biochemistry and biotechnology》1996,56(3):243-263
Papaya proteinase III (PPIII) was purified, as the S-methylthio derivative from the latex ofCarica papaya L., by ion-exchange chromatography. Separation of reactivable PPIII from the irreversibly oxidized molecular species of this
enzyme was readily achieved after a selective conversion of the reactivated proteinase into the S-monomethoxypoly-(ethylene
glycol) thio derivative (S-mPEG thio PPIII). From this derivative, a PPIII preparation titrating 1 mol of thiol/mol of enzyme
was regenerated. From the physicochemical properties of S-mPEG thio PPIII that were investigated, it is concluded that interactions
between the mPEG and the PPIII chains occur only to a limited extent. In addition to its usefulness for purifying thiol-containing
enzymes, the mPEG modification resulting from mixed disulfide bond formation may find other practical applications. 相似文献
4.
Michelle Nijs Michel Gelbcke Mohamed Azarkan Jeanne Brygier Claude Guermant Danielle Baeyens-Volant Tony Musu Claudine Paul Yvan Looze 《Applied biochemistry and biotechnology》1994,49(1):75-91
Monomethoxypoly(ethylene glycol)-N-succinimide carbonate (SC-PEG) was used to prepare PEG-lysozyme, PEG-papaya proteinase III, PEG-catalase, and PEG-lactoperoxidase
conjugates. SC-PEG produced extensively modified enzymes under mild conditions (pH 7.0; 25°C) within a couple of hours. PEG-enzyme
conjugates showed equal or even greater specific activity provided that low-molecularweight substrates were used to evaluate
the biological activities. However, papaya proteinase III and lysozyme lost their proteolytic and bacteriolytic activities,
respectively, on conjugation with PEG. This was most probably because of steric factors, since no drastic conformational changes
could be detected after conjugation of these enzymes with PEG chains. Unlike these enzymes, the secondary structures of the
two hemoproteins were somewhat affected by the covalent attachment of PEG chains as shown by FTIR experiments. These results
confirmed the potential usefulness of SC-PEG, for which a novel route of synthesis making use ofN,N′-disuccinimidyl carbonate was described. 相似文献
5.
Jeanne Brygier Jean Vlncentelli Michelle Nljs Claude Guermant Claudine Paul Danielle Baeyens-Volant Yvan Looze 《Applied biochemistry and biotechnology》1994,47(1):1-10
The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which
an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro
as pepstatin itself, however, with a 400 times higher apparent Ki. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase
Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases. 相似文献
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