Biomedical Engineering Reference
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low ratios of the BT-HAase concentration over the HA concentration the initial hydro-
lysis rates became close to zero. Under the same ionic strength condition and with the
same BT-HAase concentration, the initial hydrolysis rate could be much higher to the
condition that the HA concentration used was lower, so that the ratio of the BT-HAase
concentration over the HA concentration was higher (Figure 4). When HAase was
assayed in purified preparations, the presence of added proteins such as BSA allowed
the formation of electrostatic complexes between HA and the added protein and thus,
prevented electrostatic HA-HAase complex formation. On the contrary, when HAase
is assayed in biological samples, addition of proteins to the reaction mixture may be
not appropriated. In such cases, the total protein content may be high enough for HA
to be saturated by proteins, so that HA b(1,4) bonds are not accessible to HAase. Thus,
although HAase is present, HAase activity may be not detected in reaction mixtures
containing too high levels of proteins.
Concerning the characterization of the HAase activity, a matter of interest is that
of the influence of pH. Indeed, a great diversity of pH-dependence curves of HAase
activity were published. Considering a given HAase, as for example, BT-HAase, there
are two main reasons for this diversity. The first one concerns the experimental meth-
ods used to assay HAase activity. Indeed various methods were used to study the ef-
fect of pH on the HAase activity and, using exactly the same experimental conditions,
different pH-dependence curves can be obtained. For example, Hofinger et al. (2008)
found a maximum BT-HAase activity at pH 3.5 when using the N-acetyl-D-glucos-
amine reducing end assay whereas it was around 5.5-6 with a turbid metric method.
The second reason is that substrate-dependence curves were obtained by using a broad
range of experimental conditions in terms of ratio of the HAase concentration over the
HA concentration, purity of the HAase preparation, ionic strength and added species
(buffer, BSA, …). According to our above-mentioned results, all these experimental
conditions are precisely important with respect to the HAase activity and also to the
HA and HAase abilities to form electrostatic complexes. During our study of the pH-
dependence of the BT-HAase activity, we performed HA enzymatic hydrolysis experi-
ments in the presence of a low ionic strength (5 mmol l
-1
) by adding either BSA or LYS
at various concentrations into the reaction medium. The initial hydrolysis rates we
determined from the kinetic curves are reported on
Figures 11
and
12
(Lenormand et
al., 2010a). We can observe that in the absence of non-catalytic protein, the BT-HAase
activity was detectable only between pH 3.75 and 4.75, whereas, in the presence of
BSA (Figure 11), it was detectable at least between pH 3.5 and 5.25 and, in the pres-
ence of LYS (Figure 12), BT-HAase activity could be detected in a pH domain ranging
at least from 3 to 9. These results allow to explain those obtained by Maingonnat et
al. (1999) when they studied the activity of a tumoral HAase produced by a cell line
derived from a brain metastasis. They observed that, whatever the pH between 3.3 and
4.4, the tumoral HAase activity was strongly increased in the presence of either BSA
or an HA binding protein. Similarly, our results on Figure 11 are in total agreement
with those of Gacesa et al. (1981) according which, in a pH domain ranging from 3 to
5, BT-HAase activity was strongly increased by addition of human serum or albumin
to the reaction mixtures. Moreover, results on Figure 4 make it possible to explain the
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