The differential stability of the left and right domains of papain

We have investigated the differential stability of the two domains of papain, a broad specific cysteine protease, which is one of the most commonly used enzyme in various industries. The denaturant induced equilibrium unfolding of this enzyme has been investigated by different spectroscopic techniques. By site specific fluorescent labeling of one of the domain, we observed that during the unfolding process, L domain unfolds first and the R domain unfolds at a later stage. Spectroscopic studies reveal a biphasic unfolding transition, suggesting the presence of an intermediate during the unfolding process. The intermediate is observed between 1.5 and 2.5 M GuHCl and between 3 and 5 M in the case of urea induced unfolding. The unfolding process for both native to intermediate and intermediate to unfolded species is reversible in the case of urea unfolding, with a ΔG of ?2.4 and ?5.5 kcal/mole respectively where as for GuHCl unfolding only native to intermediate species is reversible indicating the predominance of hydrophobic interactions in the stability of the molecule.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Revealing a Concealed Intermediate that Forms after the Rate-limiting Step of Refolding of the SH3 Domain of PI3 Kinase

Kinetic and equilibrium studies of the folding and unfolding of the SH3 domain of the PI3 kinase, have been used to identify a folding intermediate that forms after the rate-limiting step on the folding pathway. Folding and unfolding, in urea as well as in guanidine hydrochloride (GdnHCl), were studied by monitoring changes in the intrinsic fluorescence or in the far-UV circular dichroism (CD) of the protein. The two probes yield non-coincident equilibrium transitions for unfolding in urea, indicating that an intermediate, I, exists in equilibrium with native (N) and unfolded (U) protein, during unfolding. Hence, the equilibrium unfolding data were analyzed according to a three-state N ↔ I ↔ U mechanism. An intermediate is observed also in kinetic unfolding studies, and its presence leads to the unfolding reaction in urea as well as in GdnHCl, occurring in two steps. The fast step is complete within the initial 11 ms of unfolding and manifests itself in a burst phase change in fluorescence. At high concentrations of GdnHCl, the entire change in fluorescence during unfolding occurs during the 11 ms burst phase. CD measurements indicate, however, that I retains N-like secondary structure. An analysis of the kinetic and thermodynamic data, according to a minimal three-state N ↔ I ↔ U mechanism, positions I after the rate-limiting transition state, TS1, of folding, on the reaction coordinate of folding in GdnHCl. Hence, I is not revealed when folding is commenced from U, regardless of the nature of the probe used to follow the folding reaction. Interrupted unfolding experiments, in which the protein is unfolded transiently in GdnHCl for various lengths of time before being refolded, showed that I refolds to N much faster than does U, confirms the analysis of the direct folding and unfolding experiments, that I is formed after the rate-limiting step of refolding in GdnHCl.     

Protein stabilizing potential of simulated honey sugar cocktail under various denaturation conditions

Protein stabilizing potential of simulated honey sugar cocktail (SHSC) against chemical and thermal denaturations was studied using bovine serum albumin (BSA) as the model protein. The two-step, three-state transition of urea denaturation of BSA became a single-step, two-state transition along with the shift in the whole transition curve towards higher urea concentrations in the presence of increasing SHSC concentrations [8–20% (w/v)] as revealed by far-UV CD, fluorescence and UV difference spectroscopic results. Far-UV and near-UV CD spectra, UV difference spectra, ANS fluorescence and three-dimensional fluorescence results suggested significant retention of native-like conformation in 4.6 M urea-denatured BSA in the presence of 20% (w/v) SHSC. A significant shift was also noticed in thermal and GdnHCl denaturation curves of BSA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested significant stabilization of BSA against urea, GdnHCl and thermal denaturations by SHSC.     

Activation of human biliverdin-IXα reductase by urea: Generation of kinetically distinct forms during the unfolding pathway

Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3 M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3 M urea, the V_max is increased 11-fold to 1.8 μmol/min/mg and the apparent K_m for biliverdin increases from 1 to 3 μM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent V_max was shown to decay to half that seen at zero time with a half life of 5.8 minutes, while the apparent K_m for NADPH remains constant at approximately 5 μM. With NADH as cofactor the half life of the activated (A) form was 2.9 minutes, and this form decays in 3 M urea to a less active (LA) form. The apparent K_m for NADH increases from 0.33 mM to 2 mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.     

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5–8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2 h, followed by adjustment to a pH in the range of 6.5–8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.     

Room temperature phosphorescence of amorphous aggregates and amyloid fibrils resulting from protein misfolding

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid fibrils.     

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.     

Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Folding and aggregation of a multi-domain engineered immunotoxin

Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (V_H/V_L) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and V_H/V_L domains had considerably different unfolding free energies (ΔG_UNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the V_H/V_L folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the V_H/V_L domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the V_H/V_L domain played a key role in aggregation of this antibody fusion protein.     

Role of the Ca-pectates on the accumulation of heavy metals in the root apoplasm

In order to better understand the processes that regulate the accumulation in the apoplasm of heavy metals and their mobilization by the plant metabolites it is essential to study the mechanisms that regulate the interactions between metal ions and pectins. In such a context, the sorption of Cd(II), Zn(II), Cu(II) and Pb(II) from single and multi-metal solutions, by a Ca-polygalacturonate gel with a degree of esterification of 18.0 (PGAM₁) and 65.5% (PGAM₂) was studied in the 3.0–6.0 pH range in the presence of CaCl₂ 2.5 mM. The sorption of Cr(III) from single metal solution was also considered. The results show that the amount of each metal ion sorbed increases with increasing the initial metal ion concentration and pH. The data from the single metal solution tests show that at pH 6.0 the affinity of the metal ions towards the PGAM₁ matrix follows the order: Cr(III) > Cu(II) ? Pb(II) ? Zn(II) ? Cd(II). The simultaneous sorption of the bivalent metal ions by the PGAM₁ gels indicates that Pb(II) is selectively sorbed. The FT-IR spectra show that the carboxylate groups are mainly responsible for the metal ion coordination. The ability of PGAM₂ to accumulate Cr(III), Cu(II), and Pb(II) was lower than that found in the PGAM₁ systems whereas the sorption of Zn(II) and Cd(II) was negligible.     

The effect of inclusion of a high lactose supplement in finishing diets on nutrient digestibility,nitrogen excretion,volatile fatty acid concentrations and ammonia emission from boars

An experiment (complete randomised design) was conducted to investigate the use of lactose in finishing pig diets on nitrogen excretion patterns and ammonia emissions. Diets were formulated to have similar digestible energy (13.8 MJ/kg) and total lysine (11.0 g/kg) contents. Thirty boars (58 kg) were assigned to one of the five dietary treatments (six per treatment) as follows: (T1) 0 g Lactofeed/kg (860 g whey permeate/kg, 140 g soya bean meal/kg, Volac International, UK) (LF70); (T2) 40 g LF70/kg; (T3) 80 g LF70/kg; (T4) 120 g LF70/kg; and (T5) 160 g LF70/kg. After a 14-day adaptation period, pigs were housed in metabolism crates and faeces and urine were collected for nitrogen balance and ammonia emission. NH₃–N emission was measured over 10 days using a laboratory scale procedure. Coefficient of total tract apparent digestibility (CTTAD) for dry matter and gross energy digestibility were affected by dietary LF70 inclusion (quadratic, P<0.05). T1 had a higher urinary N and total N excretion (P<0.1), a lower N retention (as a proportion of intake) (P<0.05) than the LF70 supplemented diets. T1 had a higher NH₃–N per gram of N intake from days 0 to 4 (P<0.05) than the LF70 supplemented diets. In the faeces isobutyric acid and isovaleric acid were affected by dietary LF70 level (quadratic, P<0.01). There was a linear decrease in the ratio of acetic acid: propionic acid with increasing LF70 levels (P<0.05). In the caecum and colon lactobacilli concentrations were affected by dietary LF70 inclusion (quadratic, P<0.05). In conclusion, the current study demonstrates that the incorporation of lactofeed in finisher pig diets increases CTTAD for gross energy and the concentration of lactobacilli in the hind-gut and decreases total nitrogen excretion. The inclusion of lactofeed also reduces the quantity of ammonia emitted from fresh manure in the first 4 days of storage.     

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k_a increased 20-fold when the concentration of urea was raised from 0 M to 6 M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the k_a value reached its maximum (k_a,max) in all urea concentrations. Interestingly, the k_a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.     

Alterations in kidney tissue following zinc supplementation to STZ-induced diabetic rats

Diabetes mellitus is a chronic disease characterized by anomalies forming in carbohydrate, lipid, protein metabolisms and the incidence of this disease varies widely throughout the world. Zinc is an important element which is essential for life and is present in nature. In this study, the animals were divided into four groups. These groups were named as untreated; zinc sulfate; streptozotocin (STZ); STZ and zinc sulfate. STZ (65 mg/kg) was dissolved in a freshly prepared 0.01 M pH 4.5 citrate buffer and given with intraperitoneal injection in a single dose. Zinc sulfate (100 mg/kg) was dissolved in distilled water and given to the animals by gavage at a daily dose for 60 days. The rats were sacrificed under ether anesthesia. This study was aimed to investigate histological and biochemical changes of zinc supplementation on the kidney tissue in STZ-induced diabetic rats. In the current study, histological and histochemical observations showed that the occurred degenerative changes decreased after giving zinc in the kidney tissue of diabetic group. Kidney glutathione (GSH) levels decreased and lipid peroxidation (LPO), nonenzymatic glycosylation (NEG), urea and creatinine levels increased in diabetic rats. GSH levels increased, while LPO, NEG, urea and creatinine levels decreased in the kidney with administration of zinc to diabetic rats. As a result, we observed curative effects of zinc given to diabetic rats. We can say that zinc may be an important antioxidant for the treatment of secondary complications of diabetes in kidney tissue.     

Self-interaction of native and denatured lysozyme in the presence of osmolytes,l-arginine and guanidine hydrochloride

Osmolyte molecules such as betaine and trehalose are protein stabilizers while l-arginine (Arg) and guanidine hydrochloride (GdnHCl) are the most widely used aggregation suppressor in protein refolding. We have herein studied the effects of the osmolyte molecules and l-arginine together with GdnHCl (0–6 mol/L) on the intermolecular interaction of native and denatured lysozyme by self-interaction chromatography. The self-interaction is characterized in terms of the osmotic second virial coefficient (B) of the protein, the increase of which represents the decrease of intermolecular attraction of the protein. It is found that the effect of Arg on the self-interaction of lysozyme is similar with GdnHCl, but its competence is much weaker than the denaturant. At higher GdnHCl concentrations (>0.5 mol/L), Arg can be used to suppress the self-association of lysozyme. In contrast to Arg, B increases with increasing betaine or trehalose concentration at the GdnHCl concentration range studied. The results indicate the cooperativity of each osmolyte with GdnHCl, and the different mechanisms of their effects from Arg on the B values. The work confirms that the osmolytes are not only protein stabilizers, but also protein aggregation suppressors for both native and denatured protein molecules.     

Effects of fibre level and dietary mannanoligosaccharides on digestibility,caecal volatile fatty acids and performances of growing rabbits

The current experiment with 3 trials aimed to study the effect of two levels of dietary fibre – high fibre (HF; 323 g aNDFom/kg) and low fibre (LF; 248 g aNDFom/kg) – and the effect of mannanoligosaccharides (MOS) addition (1 g/kg) to the LF diet (LFM) on the performances and health status of growing rabbits, digestibility and caecal fermentative characteristics. In the growth trial 132 rabbits of both sexes were used (11 cages with 4 rabbits per treatment) from weaning (32 days of age) to slaughter (67 days of age). Rabbits fed HF diet showed a significantly higher weight gain and live weight at 67 days than rabbits fed LF diet (2032 g vs. 1935 g) (P<0.05). Feed and digestible energy intake increased with dietary fibre level (P<0.05). During the growing period rabbits fed HF diet had a feed intake 26% higher than those fed LF diet. Feed efficiency ratio was worse in HF animals (0.334 vs. 0.385; P<0.05). Addition of MOS to LF diet did not affect growth performance parameters (P>0.05). Mortality and morbidity rate were not affected by treatments. In the digestibility 24 rabbits from 46 to 51 days of age trial were used. The HF diet resulted in a significant (P<0.05) decrease in digestibility of dry matter, organic matter and protein while the aNDFom digestibility was not significantly different between diets (P>0.05). Supplementation with MOS had no effects on digestibility (P>0.05). In the 3rd trial the caecal traits were measured in 30 rabbits with 46 days of age that received the experimental diets in the previous 14 days. Caecal production of total volatile fatty acids (VFA), acetate and propionate were significantly higher (P>0.05) on rabbits fed HF diets than on rabbits fed LF diets. The total VFA concentration increased 64% (from 5.01 to 8.20 mmol/100 ml) and acetate increased 73% (from 3.73 to 6.44 mmol/100 ml). Butyrate production was not different between diets (P>0.05). Fibre level did not affect proportions of VFA and caecal contents and caecal weights. Addition of MOS to LF diet did not affect any caecal trait (P>0.05). It was concluded that the reduction of dietary fibre level increases feed digestibility but worsens rabbit growth performances. Supplementation of low fibre diet with 1 g MOS/kg is not enough to reduce its negative effects on growth performances.