Philippe Dekeyser

Fractionation and purification of the thiol proteinases from papaya latex

Three cysteine proteinases, i.e. chymopapain, papaya proteinase IV and proteinase III, were purified to homogeneity from papaya latex using a combination of ion-exchange chromatography and hydrophobic interaction chromatography. During the purification procedure, the thiol-groups of the active center were reversibly blocked as mixed disulfides with 2-thiopyridone. Homogeneity was proved electrophoretically by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and rechromatography on a Mono S 5/5 column at pH 5.0.

Stabilization of fully active chymopapain by lyophilization

Abstract The use of maltodextrins, partially hydrolysed starches, was evaluated for the stabilization of fully activated chymopapain, a cysteine proteinase from the papaya latex. Chymopapain was fully activated with cysteine and Na2EDTA. Enzyme activity was determined before and after the stabilization of chymopapain by lyophilization, in the presence of different amounts of lyoprotectants. During 3 years, the stability of one of the formulations was evaluated. Differential Scanning Calorimetry (DSC) was used to determine the shelf temperature of these products, while X-ray diffraction was used to determine the crystallinity of the products. The water content was determined using Karl-Fisher titration. Maltodextrins were found to protect the fully active chymopapain during the lyophilization process. The protection was dependent on their dextrose equivalent (DE) value and independent on the concentration used (2–5% w/v). The degree of protection was as good as that obtained by sucrose. The activity of chymopapain, stabilized by maltodextrin DE 28, was constant when stored for 3 years at room temperature. Freeze-dried cakes of maltodextrin formulations, containing small amounts of cysteine and Na2EDTA, were amorphous. Maltodextrins can be considered as potential lyoprotectants in the lyophilization of fully active cysteine proteinases.

Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis

The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis.

Control of Nausea and Vomiting By Navoban(r) (tropisetron) in 131 Children Receiving Cytotoxic Chemotherapy

One hundred and thirty-one children with a median age of 5 years were administered Navoban (tropisetron), a selective antagonist of the serotonin receptor (5-HT3), dosed once daily at 0.2 mg/kg (with a maximum of 5 mg daily) in a study aimed at evaluating the prevention of nausea and vomiting induced by anti-cancer chemotherapy. The most common malignancy (in 49% of patients) was acute lymphocytic leukaemia. Patients received Navoban during one or more courses of emetogenic chemotherapy for a total of 455 courses administered intravenously or intravenously and intrathecally (IV + IT). Most patients (89%) had already received cytotoxic chemotherapy before enrollment for the trial. On Day 1, Navoban was administered slowly and intravenously as a single dose before the start of chemotherapy, or by mouth as a single daily dose on subsequent days (median treatment duration = 5 days). On the first 5 days of each course of chemotherapy, response to Navoban per 24-hour period was graded as: complete (absence of both nausea and vomiting), partial (1-4 vomits and/or less than 5 hours of nausea), or failure (more than 4 vomits and/or at least 5 hours of nausea). Ninety-six per cent of the intravenous chemotherapy group and 97% of the IV + IT chemotherapy group had a complete (70% and 55% respectively) or partial (26% and 42% respectively) response during the first 24-hour period of the first course in which Navoban was used. The second and subsequent courses yielded similar percentages. Delayed emesis was observed mainly during those courses employing the most emetogenic chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance size-exclusion chromatography of proteoglycans extracted from bovine articular cartilage

Abstract The applicability of high-performance size-exclusion chromatography for determining the relative molecular weight of proteoglycans was tested with a new type of column. The method is extremely reproducible, precise and rapid and allows molecular weight determinations up to 3 millions, even done in the presence of considerable impurities of low molecular weight. This technique offers important advantages over the traditional techniques, such as light scattering, sedementation velocity and equilibrium ultracentrifugation and viscosity. The peak position method was used to calibrate our system using pullulan standards. For the pullulan standards in the molecular weight range between 1.0 × 10 4 and 8.5 × 10 5 , an almost linear relationship between log M p and the ratio between the retention time of the standard and that of the excluded salt was found. For the lower molecular weight standards, even at the lowest measured concentration, the curve deviated sharply for ratios above 0.80. The influence of the flow-rate on this ratio was negligible. We were able to separate the proteoglycan aggregates from the proteoglycan monomers in the KCl extracts. In the CaCl 2 extract however, we could not separate the monomers from the aggregates, since these were present in a much higher concentration than in the KCl extract. After the addition of hyaluronidase from bovine testes, the proteoglycan aggregates were broken down by lysis of the hyaluronic acid molecules, who are essential for the formation of these aggregates.