Biomedical Engineering Reference
In-Depth Information
In the course of the last 10 years, we have studied the kinetics of the HA hydro-
lysis catalyzed by HAase. An atypical kinetic behavior was observed whose extent
varied according to the experimental conditions used. We then showed that, in fact,
to properly describe the behavior of the HA/HAase system, the ability of HA to form
electrostatic complexes with proteins, including HAase, should be taken into account
in addition to the formation of the HA-HAase catalytic complexes. Our studies also
showed that, as a consequence, the HAase activity towards HA can be strongly modu-
lated by the formation of electrostatic complexes involving either HA or HAase or
both. In addition to review these results and their main conclusions, we discuss here
their relevance under both
in vitro
and
in vivo
situations.
ha aNd haase: CatalytiC ComPleXes Versus eleCtrostatiC
ComPleXes
For our study of the kinetics of the HA hydrolysis catalyzed by HAase, we used bovine
testicular HAase (BT-HAase), which is commercially available, as a model enzyme.
Indeed, this enzyme is the bovine counterpart of the human PH-20 and, like all the
human HAases, it is of the testicular type. In addition, it was shown that the human
HAases share 33.1-41.2% aminoacid sequence identities and a higher degree of struc-
tural similarity (Chao et al., 2007; Jedrzejas and Stern, 2005). Among the methods
used to study the HA hydrolysis catalyzed by HAase, we chose the colorimetric assay
for N-acetyl-D-glucosamine reducing ends, also known as the Reissig method (Reissig
et al., 1955) or the Morgan-Elson method (Elson and Morgan, 1933), because it is the
only one for which the measurement is directly related to the number of b(1,4) bonds
cleaved. Indeed, each hydrolysis of a b(1,4) bond leads to a one unit increase in the
number of HA chains and so, the same for the number of N-acetyl-D-glucosamine
reducing ends
(Figure 2).
We used our improved version of the method which allows
to deduce the turbidimetric contribution from the total absorbance measured at 585
nm and thus, to obtain the actual colorimetric contribution. This colorimetric compo-
nent of the absorbance at 585 nm is proportional to the concentration of N-acetyl-D-
glucosamine reducing ends in the sample (Asteriou et al., 2001). Thus, for each kinetic
experiment, the concentration of N-acetyl-D-glucosamine reducing ends was plotted
against the reaction time. The experimental points were fitted by the bi-exponential
equation we developed (Vincent et al., 2003) and the initial hydrolysis rate was cal-
culated as being equal to the value of the first derivative of that function at time zero.
Initial hydrolysis rates are thus expressed in µmol of N-acetyl-D-glucosamine reduc-
ing ends released per liter of reaction medium and per minute.
Our study of the influence of the HA concentration on the initial rate of the HA
hydrolysis catalyzed by BT-HAase under low ionic strength conditions (5 mmol l
-1
)
revealed an atypical behavior
(Figure 3)
for increasing HA concentrations, the initial
hydrolysis rate successively increased, reached a maximum and then decreased to a
very low level, close to zero, at high HA concentration, instead of reaching a plateau,
as it does for a Michaelis-Menten type enzyme (Asteriou et al., 2002, 2006). The
same atypical behavior was observed whatever the BT-HAase concentration used
(Figure 3).
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