Christine Robert

Inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase by carboxylic acids

Abstract The inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is reported. Recently, two forms of palmito PPO were partially purified by hydrophobic chromatography. Inhibitory effects of various carboxylic acids on these two forms have been studied. Both forms showed identical behaviour towards the inhibitors studied. Cinnamic acid was found to have the greatest inhibitory effect (Ki = 0·06 mM). When the inhibitory effects of acids from the benzoic acid family and from the cinnamic acid family were compared, it was found that acids which possess a double bond between the benzene ring and the carboxylic function showed the highest inhibitory effect. This inhibitory effect was decreased by substitutions on the benzene ring. The influence of pH on the inhibitory effect of carboxylic acids on palmito PPO has also been investigated. Ki decreased with a decrease in pH. This effect is due to the fact that it is only the undissociated form (AH) of carboxylic acids that is responsible for inhibition of PPO. Inhibition constants (Ki for the AH form have been recalculated and were found to remain constant in the pH range studied (for benzoic acid Ki = 0·14 mM, for cinnamic acid Ki = 0·019 mM, for sorbic acid Ki = 0·15 mM).

Simultaneous Determination of Sugars by Multivariate Analysis Applied to Mid-Infrared Spectra of Biological Samples

We have investigated the use of principal component analysis (PCA) to describe and assess mid-infrared spectral data obtained from complex biological samples containing sucrose, fructose, and glucose. The correlation coefficients between spectral data and chemical values of each variable (sucrose, glucose, fructose, total sugars, and reducing sugars) showed that in each case, axes 1, 3, 4, and 5 had the highest values. These values also indicated which axes each variable was mostly correlated with. The results also showed that the samples were distributed according to their sucrose concentrations (or total sugars) along a concentration gradient in the projection plan formed between axes 1 and 3. No clear discrimination according to concentration was observed with other factorial maps. Prediction equations that linked sucrose, fructose, glucose, total sugar, and reducing sugars concentrations to the spectral data were established by regression on the principal component. Very high correlation coefficients values between the first 10 axes and the chemical values were obtained (between 0.9757 and 0.998). From such aqueous biological samples containing a ternary mixture of sucrose, fructose, and glucose, it was possible to (1) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars/total sugars) and (2) to specifically measure their concentrations with a relatively good accuracy.

A kinetic study of the inhibition of palmito polyphenol oxidase by L-cysteine

Abstract Polyphenol oxidase (PPO) is responsible for browning reactions in fruits and vegetables. In order to inhibit these reactions during processing, the use of chemical inhibitors including thiols such as L-cysteine has been proposed. The effect of this thiol on PPO activity was studied in order to establish if it reacts with quinone and/or directly inhibits the enzyme. The inhibition of palmito PPO (catecholase activity, with 4-methylcatechol as substrate) by L-cysteine was characterized spectrophotometrically. The effect of increasing cysteine concentration with respect to incubation time was measured by polarography and the kinetic parameters calculated. Kinetic analysis using spectrophotometric assays showed a lag period suggesting that there is no quinone accumulation in the reaction mixture. The increase of absorbance at 300 nm observed with u.v.—vis spectra indicated that a colourless compound (thiol—quinone complex) was produced. The plots of log remaining enzyme activity vs incubation time were characterized by two straight lines suggesting two possible inactivation models: (i) the deactivation of one enzyme that proceeds in two steps or (ii) the existence of two isoenzymes that behave differently. We have shown that the inactivation process of PPO by cysteine followed this model: N → X[C] → I where N represents one native form, X represents an intermediate form, the structure of which depends on the cysteine concentration [C], and where I is the completely inactive form of the enzyme. These results showed that there is a direct irreversible inhibition of PPO by cysteine according to a two-step model. The analytical approach illustrated by the present study of palmito PPO inhibition by L-cysteine may be applied to other investigations of mechanisms of enzyme inhibition.

Enzyme kinetics by mid-infrared spectroscopy: β-fructosidase study by a one-step assay

An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.

ANALYSIS OF NEAR-INFRARED SPECTRA OF SOME CARBOHYDRATES

Abstract Infrared, in particular near-infrared (NIR) is a very useful technique for the identification and the characterization of biomolecules. The complexity of the spectra tends to make direct interpretation difficult. However, from the spectroscopic representation of the factorial axes obtained by principal component analysis (PCA) performed on different sugar molecules, it is possible to determine the characteristic wavelengths (NIR) of monosaccharides, oligosaccharides and polysaccharides. The most characteristic wavelengths for monosaccharides are found to be: 1457 nm, 2062 nm, 2263 nm and 2440 nm; and for oligo- and polysaccharides: 1432 nm, 1931 nm, 2170 MI, 2310 nm and 2477 nm. These wavelengths, which have been identified from the spectral profiles, are the most appropriate for a quantitative study of unknown carbohydrate mixtures.

Assessment of the C4 phosphoenolpyruvate carboxylase gene diversity in grasses (Poaceae)

Abstract.C4 phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway. To analyze the diversity of the corresponding gene in grasses, we designed PCR primers to specifically amplify C4 PEPC cDNA fragments. Using RT-PCR, we generated partial PEPC cDNA sequences in several grasses displaying a C4 photosynthetic pathway. All these sequences displayed a high homology (78–99%) with known grass C4 PEPCs. PCR amplification did not occur in two grasses that display the C3 photosynthetic pathway, and therefore we assumed that all generated sequences corresponded to C4 PEPC transcripts. Based on one large cDNA segment, phylogenetic reconstruction enabled us to assess the relationships between 22 grass species belonging to the subfamilies Panicoideae, Arundinoideae and Chloridoideae. The phylogenetic relationships between species deduced from C4 PEPC sequences were similar to those deduced from other molecular data. The sequence evolution of the C4 PEPC isoform was faster than in the other PEPC isoforms. Finally, the utility of the C4 PEPC gene phylogeny to study the evolution of C4 photosynthesis in grasses is discussed.

Inhibition of Palmito Polyphenoloxidase by Halide Salts

The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller F- ion is a stronger inhibitor than I- and Br- and that Cl- has the highest Ki value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.

A new human transporter associated with antigen processing alleles encodes a large C-terminal protein domain.

The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. Five nucleotide variation sites that induce a change in amino acid residues were identified in the TAP2 gene, and seven alleles derived from the combination of these dimorphic sites have been described (Bahram et al. 1991; Colona et al. 1992; Moins-Teisserenc et al. 1994; Szaffer et al. 1994; see Table 1). In the present study, TAP2 typing was performed by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) as previously described (Powis et al. 1993). Oligonucleotide primers used for this typing are shown in Table 2. A group of 78 families living on Reunion Island, consisting of 82 insulin-dependent diabetes mellitus (IDDM) patients and most of their first-degree relatives (i. e., 154 parents and full sibling subjects), were studied. Seventeen of the 236 persons analyzed (seven IDDM patients and ten parental controls) showed a new combination of nucleotides located at polymorphic sites, allowing the definition of a new allele. This new allele, TAP2 G (see Table 1), was characterized by the presence of amino acid residues Thr on position 665 and Gln on position 687. No exception to the previously detected association between 655 Ala and 687 Gln or 665 Thr and 687 Stop has been described in Caucasians (Martinez-Laso et al. 1994; Szaffer et al. 1994). This linkage disequilibrium which was considered absolute in Caucasians (IDDM patients and control) is not recovered in the Reunion Island population, which is highly crossbred (including Caucasians, Africans, Indians, and Chinese). Sequence analysis of cDNA PCR amplified fragment, coming from the homozygous immortalized B-cell line Reu-M4-1, has confirmed the existence of this allele.

Microquantification of Proteins by Spectrophotometry. Part II : Application Procedure for Complex Mixture Containing Interfering Substances

Abstract In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developped a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99 % yield, and thus making microquantification possible. This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64 % res...