Danielle Baeyens-Volant

Statistical theories of main chain scission and crosslinking of polymers—application to the photolysis and radiolysis of polystyrene studied by gel permeation chromatography

Abstract The statistical theories of main chain scission and crosslinking resulting from either homogeneous or inhomogeneous energy dissipation in polymers are summarized. The molecular weight distributions of polystyrene samples subjected in vacuo to y-rays (homogeneous energy dissipation) or to U.V. irradiation (inhomogeneous energy dissipation) have been measured by GPC for various absorbed doses. The results are discussed according to these theories.

A new family of liquid crystals: N-substituted aldonamides. II: Relationship between chemical structure and the formation of mesophases

Abstract A series of N-substituted aldonamides has been synthesized. Some of these compounds form a thermotropic lamellar mesophase in which molecules in the trans-configuration are arranged in monolayers stabilized by hydrogen bonding and hydrophobic interactions. X-ray diffraction studies have shown that a molecular arrangement of this type exists in the crystalline solid of N-decylribonamide. The relationship between the mesogenic properties and the molecular structures of these compounds is discussed (with reference to the length, branching and cyclisation of the aldonic residue and of the hydrocarbon chain).

Reversible modification of thiol-containing polypeptides with poly (ethylene glycol) through formation of mixed disulfide bonds. The case of papaya proteinase III.

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.

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

Background: The Kunitz-STI family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general. Results: PPI is a versatile protease inhibitor that targets several subfamilies of serine proteases. Conclusion: The β-trefoil fold constitutes an evolutionary platform for protease inhibition and molecular recognition. Significance: Fold plasticity influences protein evolution toward multiple function and binding promiscuity. Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.

Preparation and characterization of a S-monomethoxypoly-(ethylene glycol) thioderivative of papain

Abstract A derivative of papain in which the free thiol group of the proteinase is attached to a poly(ethylene glycol) chain via a disulphide bond has been prepared. The poly(ethylene glycol) chain confers to the conjugate new physico-chemical properties, including a looser binding to ion exchange chromatographic supports such as CM-Sephadex C-50. The conjugate is thus readily separated from the irreversibly oxidized (unconjugated) forms of the enzyme. A papain preparation titrating 0.97 mol of thiol per mol of enzyme is obtained from the conjugate.

Covalent attachment of poly(ethyleneglycol) to peptides and proteins - Reevaluation of the synthesis, properties, and usefulness of carbonylimidazol-1-yl-methoxypoly(ethyleneglycol)

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.

N-Substituted aldonamides: III. Relationship between chemical structure and the formation of lyotropic mesophases

Abstract The lyotropic behavior of a series of N-substituted aldonamides which have both lyotropic and thermotropic characteristics, is systematically investigated as a function of the length, branching, and cyclization of the aldonic residue and of the alkyl chain. The ability of these compounds to form lyotropic phases is related to their chemical structure. The structures of the lamellar thermotropic and lyotropic phases are compared, and the continuity between both phases is presented.

Natural Mercury Levels in Geological Enriched and Geological Active Areas: Case Study of Katun River and Lake Teletskoye, Altai (Siberia)

Natural geological Hg deposits control the Hg levels inthe upper Katun river. Very high levels of total Hg areobserved in the watercolumn (up to 20 ng L-1) and thesediments (up to 244 μg g-1) close to the depositarea, but almost normal levels (1.8 ng L-1 in the watercolumn and 0.14 μg g-1 in the sediments) are reached60 km downstream of that zone. In general, low dissolvedmethylmercury (MMHg) concentrations were found (0.04–0.05 ngL-1) due to unfavourable methylation conditions. The MMHgconcentrations in the sediments vary from 23.3 ng g-1, inthe vicinity of the geological Hg deposits, to 0.17 ng g-1 60 km downstream.Total Hg levels in Lake Teletskoye (a geological activearea) are slightly increased (1.1–1.8 ng L-1) compared toLake Baikal and fairly constant alover the Lake, suggestingmultiple sources. High mercury concentrations in springs andsoils coincide with high radon concentrations in the samecompartments as well as high soil exhalation fluxes. Theseresults in combination with the fact that Lake Teletskoye islocated in an active fault zone suggest that the Rn and Hgsources may be fault aligned spring waters and deep seatedgases escaping through open cracks. Methylmercuryconcentrations in the Lake (0.03–0.1 ng L-1) werecomparable to the concentrations found in Katun river butrelative to the total Hg burden this means a higher percentage.

A novel form of ficin from Ficus carica latex: Purification and characterization

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS-PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS-PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% β-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg(2+), Ca(2+) and Mn(2+) at a concentration up to 10mM. However, the activity was completely suppressed by Zn(2+) at a concentration of 1mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS-PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation.