Biomedical Engineering Reference
In-Depth Information
which HA and BT-HAase are able to form electrostatic complexes different from the
catalytic complexes.
The fact that each glucuronic acid residue bears a carboxyl group makes HA chains
behave as polyelectrolytes whose pK
0
was estimated to be equal to 2.9 (Berriaud
et al., 1998; Cleland et al., 1982). This means that, except under very acidic pH condi-
tions, HA is a polyanion. Thus, according to the ionic strength level, HA may be able
to form electrostatic complexes with polycations. In fact, the ability of HA to form
electrostatic complexes with proteins was shown more than 60 years ago. Indeed,
the very first methods developed to assay HAase were based on the measurement of
the turbidity resulting from the formation of complexes between long HA chains and
serum proteins under acidic conditions: the lower the turbidity, the higher the HAase
activity (Deschrevel et al., 2008a). The turbidimetric method was further developed
by Tolksdorf et al. (1949), introducing the definition of the turbidity reducing unit
based on the turbidity obtained when HA is mixed at pH 3.8 with the bovine serum
albumin (BSA). This method still constitutes the current United States Pharmacopeia
XXII assay for HAase. More recently, investigations (Gold, 1980; Grymonpré et al.,
2001; Malay et al., 2007; Moss et al., 1997; Van Damme et al., 1994; Xu et al., 2000)
were devoted to the characterization of the complexes formed between HA and some
proteins. For example, Xu et al. (2000) examined the influence of pH on the solubility
of the electrostatic HA-BSA complexes and Moss et al. (1997) studied the electrostatic
complexes formed between HA and lysozyme (LYS).
For our kinetic experiments, there was neither protein nor any other polycation
added into the reaction media. The only one species which could form electrostatic
complexes with HA was thus BT-HAase. By using an electrophoretic method, we
estimated the isoelectric point (pI) of BT-HAase to be close to 7, which mean that,
under acidic pH conditons, the net charge of BT-HAase is positive. Thus, HA and BT-
HAase should be able to form electrostatic complexes at least between pH 2.9 and 7.
In fact, the first time we experimentally observed the ability of HA and BT-HAase to
form electrostatic complexes under acidic pH conditions was when we investigated
the origin of the turbidity component of the total absorbance measured at 585 nm in
the N-acetyl-D-glucosamine reducing end assay (Asteriou et al., 2001). Then, by using
HA and BSA, as a model system for the formation of electrostatic HA protein com-
plexes, and by performing turbidimetric measurements, we confirmed the existence of
electrostatic HA-BT-HAase complexes under the pH conditions we used in our kinetic
studies (Deschrevel et al., 2008a; Lenormand et al., 2008). In fact, in the case of the
HA/BT-HAase system, study of the electrostatic complexes is complicated by the fact
that the enzymatic hydrolysis of HA leads to a decrease in the HA chain length and,
we showed, using the HA/BSA system, that the formation of electrostatic complexes
depends on the size of HA chains (Lenormand et al., 2010b).
The investigations we carried out in order to explain the kinetic atypical behavior
of the HA/BT-HAase system thus led us to the conclusion that, in fact, two phenom-
ena must be considered to properly describe the behavior of that system: on one hand,
the formation of catalytic complexes between HA and BT-HAase which leads to HA
hydrolysis, and, on the other hand, the formation of electrostatic complexes between
Search WWH ::
Custom Search