Cell Wall Composition of Hemp Shiv Determined by Physical and Chemical Approaches.

The use of agricultural by-products in the building engineering realm has led to an increase in insulation characteristics of biobased materials and a decrease in environmental impact. The understanding of cell wall structure is possible by the study of interactions of chemical compounds, themselves determined by common techniques like Van Soest (VS). In this study, a global method is investigated to characterise the cell wall of hemp shiv. The cell wall molecules were, at first, isolated by fractionation of biomass and then analysed by physical and chemical analysis (Thermal Gravimetric Analysis, Elementary Analysis, Dynamic Sorption Vapor and Infra-Red). This global method is an experimental way to characterise plant cell wall molecules of fractions by Thermal Gravimetric Analysis following by a mathematical method to have a detailed estimation of the cell wall composition and the interactions between plant macromolecules. The analyzed hemp shiv presents proportions of 2.5 ± 0.6% of water, 4.4 ± 0.2% of pectins, 42.6 ± 1.0% (Hemicellulose-Cellulose), 18.4 ± 1.6% (Cellulose-Hemicellulose), 29.0 ± 0.8% (Lignin-Cellulose) and 2.0 ± 0.4% of linked lignin.

Hyaluronidase activity is modulated by complexing with various polyelectrolytes including hyaluronan.

Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biological properties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst the substrate-dependence curve has a Michaelis-Menten shape with a 0.15 mol L(-1) ionic strength, at low ionic strength (5 mmol L(-1)), a strong decrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximum increases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by the ability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify this hypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albumin then was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to be free and catalytically active. The kinetic results showed that the HA-HAase non-specific complex inhibits HAase catalytic activity towards HA. Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induce another type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition or activation, in particular under in vivo conditions, is discussed.

Reaction-complexation coupling between an enzyme and its polyelectrolytic substrate: determination of the dissociation constant of the hyaluronidase-hyaluronan complex from the hyaluronidase substrate-dependence.

Hyaluronan (HA) is catalytically hydrolyzed by hyaluronidase (HAase). Depending on pH, HA is able to form a non-productive electrostatic complex with HAase in addition to the classical enzyme-substrate complex. Experiments have shown the strong inhibition of the HA hydrolysis catalyzed by HAase when performed at high HA over HAase concentration ratio and low ionic strength. The substrate-dependence thus shows a non-classic inhibition of HAase at high substrate concentrations due to the sequestration of HAase by HA in the electrostatic complex. The modeling of the HA/HAase system is characteristic of a reaction-complexation coupling and it is very difficult to study reaction or binding, separately. Here, we have established the equation controlling the global system and shown that the substrate-dependence of such a system is a direct combination of a pure Michaelis-Menten equation associated with the reaction and a hyperbolic curve associated with the binding. At low substrate concentrations, the hyperbola, representing the relative part of HAase not sequestered by HA, can be assimilated to a straight line. We have established the relationship between the slope of that straight line and the dissociation constant of the electrostatic HA-HAase complex. Fitting the theoretical equation to the experimental data allowed us to determine, for the first time, the Kd value of the non-productive HA-HAase complex at low ionic strength.

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence.

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.

How hyaluronan-protein complexes modulate the hyaluronidase activity: the model.

Hyaluronan (HA) is the substrate of hyaluronidase (HAase). In addition, HA is able to form electrostatic complexes with many proteins, including HAase. Experiments have shown the strong inhibition of the HA hydrolysis catalyzed by HAase when performed at low HAase over HA concentration ratio and under low ionic strength conditions. Non-catalytic P proteins are able to compete with HAase to form electrostatic complexes with HA and thus to modulate HAase activity. We have modeled the HA-HAase-P system by considering the competition between the two complex equilibria HA-P and HA-HAase, the Michaelis-Menten type behavior of HAase, and the non-activity of the electrostatically complexed HAase. Simulations performed by introducing experimental data produce a theoretical behavior similar to the experimental one, including all the atypical phenomena observed: substrate-dependence, enzyme-dependence and protein-dependence of HAase. This shows that our assumptions are sufficient to explain the behavior of the system and allow us to estimate unknown parameters and suggest new developments.

The hyaluronan-protein complexes at low ionic strength: how the hyaluronidase activity is controlled by the bovine serum albumin.

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at low HAase over HA concentration ratio and under low ionic strength conditions. The reason is the ability of long HA chains to form electrostatic and non-catalytic complexes with HAase. For a given HA concentration, low HAase concentrations lead to very low hydrolysis rates because all the HAase molecules are sequestered by HA, whilst high HAase concentrations lead to high hydrolysis rates because the excess of HAase molecules remains free and active. At pH 4, non-catalytic proteins like bovine serum albumin (BSA) are able to compete with HAase to form electrostatic complexes with HA, liberating HAase which recovers its catalytic activity. The general scheme for the BSA-dependency is thus characterised by four domains delimited by three noticeable points corresponding to constant BSA over HA concentration ratios. The existence of HA-protein complexes explains the atypical kinetic behaviour of the HA / HAase system. We also show that HAase recovers the Michaelis-Menten type behaviour when the HA molecule complexed with BSA in a constant complexion state, i.e. with the same BSA over HA ratio, is considered for substrate. When the ternary HA / HAase / BSA system is concerned, the stoichiometries of the HA-HAase and HA-BSA complexes are close to 10 protein molecules per HA molecule for a native HA of 1 MDa molar mass. Finally, we show that the behaviour of the system is similar at pH 5.25, although the efficiency of BSA is less.

Electrostatic interactions between hyaluronan and proteins at pH 4: how do they modulate hyaluronidase activity.

Hyaluronan (HA) hydrolysis catalyzed by hyaluronidase (HAase) is inhibited at low HAase over HA ratio and low ionic strength, because HA forms electrostatic complexes with HAase, which is unable to catalyze hydrolysis. Bovine serum albumin (BSA) was used as a model to study the HA-protein electrostatic complexes at pH 4. At low ionic strength, there is formation of (i) neutral insoluble complexes at the phase separation and (ii) small positively-charged or large negatively-charged soluble complexes whether BSA or HA is in excess. According to the ionic strength, different types of complex are formed. Assays for HA and BSA led to the determination of the stoichiometry of these complexes. HAase was also shown to form the various types of complex with HA at low ionic strength. Finally, we showed that at 0 and 150 mmol L(-1) NaCl, BSA competes with HAase in forming complexes with HA and thus induces HAase release resulting in a large increase in the hydrolysis rate. These results, in addition to data in the literature, show that HA-protein complexes, which can exist under numerous and varied conditions of pH, ionic strength and protein over HA ratio, might control the in vivo HAase activity.