Control of the Hyaluronidase Activity Towards Hyaluronan by Formation of Electrostatic Complexes: Fundamental and Application Relevance - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 133

As we have observed on Figure 3 , the initial rate of HA hydrolysis catalyzed by
BT-HAase depended on the ratio of the BT-HAase concentration over the HA concen-
tration. Indeed, the higher the ratio of the BT-HAase concentration over the HA con-
centration, the higher the concentration of active BT-HAase and the higher the initial
hydrolysis rate. Nevertheless, the ability of HA and BT-HAase to form electrostatic
complexes foremost depends on both pH and ionic strength conditions. As mentioned
above, HA and BT-HAase are able to form electrostatic complexes at least between
pH 2.9 and 7, pH domain in which simultaneously HA behaves as a polyanion and
BT-HAase as a polycation. Moreover, the higher the ionic strength level, the lower the
stability of the electrostatic HA-BT-HAase complexes. Indeed, an increase in the ionic
strength level leads to an increase in the screening of the charges borne by HA and
BT-HAase by small ions and thus, to a decrease in the ability of these two polyelec-
trolytes to form electrostatic complexes. This is in very good agreement with the fact
that the atypical behavior tended to decrease, up to disappear, when the ionic strength
of the reaction medium was increased ( Figure 4 ) . However, our experiments showed
that the formation of electrostatic complexes between HA and BT-HAase can occur
under physiological-type ionic strength since the atypical behavior was observed with
an ionic strength equal to 0.15 mol l -1 (Lenormand et al., 2008).
eNhaNCemeNt/suPPressioN oF haase aCtiVity toWards ha By
PolyCatioNs aNd PolyaNioNs
As mentioned above, various proteins are able to form electrostatic complexes with
HA. We thus decided to investigate the effect of the presence of a protein with no cata-
lytic activity towards HA on the kinetics of the HA hydrolysis catalyzed by BT-HAase.
For that purpose, we chose to first use BSA. Indeed, BSA is known as to be able to
form electrostatic complexes with HA. In addition, BSA is a major protein of synovial
fluid (Scott et al., 2000), a fluid which is also rich in HA (see Introduction section).
Figure 6 shows BSA-dependence curves (that is to say, initial hydrolysis rate plotted
as a function of the BSA concentration) obtained for various HA concentrations, at pH
4 and at low ionic strength (5 mmol l -1 ). We can observe (Figure 6) that (i) the initial
rate of HA hydrolysis catalyzed by BT-HAase strongly depended on the BSA concen-
tration and (ii) the BSA-dependence curves had all the same shape whatever the HA
concentration (Lenormand et al., 2009).
In fact, the BSA-dependence curves can be divided into four domains ( Figure 7)
(Deschrevel et al. 2008a; Lenormand et al., 2009):
• The first domain corresponds to BSA concentrations ranging from zero to A.
When the BSA concentration is nil, nearly all the BT-HAase molecules form
electrostatic complexes with HA. The concentration of catalytically active BT-
HAase is thus close to zero, which makes the initial hydrolysis rate extremely
low. For increasing BSA concentrations up to A, the added BSA molecules use
the space remaining free on HA molecules to form electrostatic complexes. This
has no effect on the initial hydrolysis rate which remains close to zero. It should
be noted however that domain 1 exists only for low values of the ratio of the
BT-HAase concentration over the HA concentration.