The hyaluronan–protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin

Matrix Biology 28 (2009) 365–372

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Matrix Biology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t b i o

The hyaluronan–protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin Hélène Lenormand, Frédéric Tranchepain, Brigitte Deschrevel, Jean-Claude Vincent ⁎ Laboratoire “Polymères, Biopolymères, Surfaces”, FRE 3101 CNRS, Université de Rouen, 76821 Mont-Saint-Aignan cedex, France

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Article history: Received 10 February 2009 Received in revised form 14 April 2009 Accepted 14 April 2009 Keywords: Hyaluronan Hyaluronidase Bovine serum albumin Polysaccharide–protein complex Complex stoichiometry Hydrolysis rate Activation/inhibition of hyaluronidase

a b s t r a c t 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. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Interactions between two biomacromolecules occur according to two main types of mechanisms: non-compatibility between the two biomacromolecules leading to thermodynamic segregation, or electrostatic interactions leading to complex coacervation (Schmitt et al., 1998). The protein–polysaccharide complexes belong to the latter group. Complexes between proteins and polyelectrolytes (Cooper et al., 2005) have essentially been used in micro-encapsulation (Palmieri et al., 1999) and protein purification (Wang et al., 1996). Proteins and polysaccharides have an important role in the organization and the functioning of living cells and we think that complex coacervation can be involved in the regulation of enzyme activities in the extracellular matrix (ECM) which contains high concentrations of both charged polysaccharides and proteins. Hyaluronan (HA) is an anionic polysaccharide, present in the ECM of connective, growing and tumour tissues and implicated in cellular adhesion, mobility and differentiation processes (Catterall, 1995; Delpech et al., 1997a; Kennedy et al., 2002; Laurent, 1987; Rooney et al., 1995; Girish and Kemparaju, 2007). It is a polysaccharide of high molar mass composed of D-glucuronic acid-β(1,3)-N-acetyl-Dglucosamine disaccharide units linked together through β(1,4) glyco⁎ Corresponding author. Tel.: +33 2 35 14 67 42; fax: +33 2 35 14 67 04. E-mail address: [email protected] (J.C. Vincent). 0945-053X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.matbio.2009.04.008

sidic bonds. HA is the substrate of hyaluronidase (HAase) which catalyses its cleavage into oligosaccharides. HA and HAase are simultaneously present at high concentrations in tumour tissues (Lokeshwar et al., 1996; Victor et al., 1997; Stern, 2004). As oligosaccharides of HA (4 to 25 disaccharides) have an angiogenic action (Rooney et al.,1995; West et al., 1985; West and Kumar, 1989) contrary to native HA (Deed et al., 1997), the balance between high molecular weight HA (HWHA) and low molecular weight HA (LWHA) may play a role in cancer development (Stern, 2008; Lokeshwar and Selzer, 2008). According to Mio and Stern (2002), regulation of the size and concentration of HA chains is kinetically and energetically more efficient through the catabolic rather than the anabolic pathway. In fact, HAase could be present in tissues together with inhibitors, which may allow HAase to be quickly activated or inactivated. The balance between LWHA and HWHA could thus be controlled by the action of HAase (Delpech et al., 1997a; Mio and Stern, 2002; Cameron, 1966) and it is of great importance to understand the kinetics of that enzyme reaction. We examined the kinetics of native HA hydrolysis catalysed by HAase (Vincent et al., 2003; Astériou et al., 2002, 2006), using bovine testicular HAase (BTHAase) as a model, since it is commercially available and, like human HAase, it catalyses the hydrolysis of the β(1,4) bonds in HA (Csóka et al., 2001). Using a physiological-type ionic strength, i.e. 0.15 mol L− 1, BTHAase at 1 g L− 1 obeys the Michaelis–Menten law: the initial reaction rate is a hyperbolic function of the substrate concentration (Vincent et al., 2003). At very low ionic strength, the behaviour of

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the reactive system is totally different. An atypical kinetic behaviour of the HA / HAase system appears: for increasing HA concentrations, the initial hydrolysis rate successively increases, reaches a maximum and then decreases to a very low level, close to zero, at high substrate concentrations, instead of reaching a plateau, as it does for a Michaelis– Menten type enzyme (Astériou et al., 2006). The conclusion was that the atypical behaviour of the system can be attributed to the existence of HA–HAase non-specific and non-catalytic electrostatic complexes (Astériou et al., 2006; Lenormand et al., 2007), due to the polyelectrolytic nature of HAase. The ability of polysaccharides and proteins to form complexes has been known for a long time and, in the case of HA, it was shown more than 60 years ago. The Mucin Clot Prevention (Robertson et al., 1940) and the turbidimetric methods (Kass and Seastone, 1944; Dorfman and Ott, 1948) have been developed to assay HAase. In the latter method, HAase is assayed by measuring the disappearance of the turbidity resulting from complexes formed between long chains of HA and albumin under acidic conditions. Since then, some investigations (Gold, 1980; Grymonpré et al., 2001) have been devoted to the characterisation of the complexes formed between HA and the bovine serum albumin (BSA). For example, Xu et al. (2000) have studied the influence of pH on the solubility of electrostatic HA–BSA complexes. Moreover, others working on HAase report that serum proteins are able to enhance the HAase catalytic activity; Gacesa et al. (1981) have shown that among the serum proteins, albumin has the greatest effect. According to Maingonnat et al. (1999), other proteins such as hyaluronectin (HN), which is a hyaladherin (i.e. a protein able to specifically interact with HA (Delpech et al., 1997b)), hemoglobin and immunoglobulins are also able to enhance the HAase activity and hence, increase the sensitivity of the HAase detection. We have shown that BSA is not only able to complex HA but also able to compete with HAase, when simultaneously present, favouring the liberation of HAase molecules which recover their catalytic activity (Lenormand et al., 2008; Deschrevel et al., 2008). The result is an enhancement of the apparent HAase activity. However, the HAase activity enhancement depends on the protein concentration. When the hydrolysis is performed at pH 4, 1 g L− 1 HA concentration and 0.5 g L− 1 HAase concentration, the initial hydrolysis rate increases at low BSA concentration, reaches a maximum for a BSA concentration equal to 0.65 g L− 1, then decreases at high BSA concentrations (Lenormand et al., 2008; Deschrevel et al., 2008). That means that an optimum BSA concentration exists. Maingonnat et al. (1999) reported a dependency of the HAase activity with respect to the concentration of proteins, BSA as well as hyaluronectin, at pH 3.8 which shows that a protein concentration ranging from 0.02 to 0.2 is optimal to enhance the HAase activity, whatever the HAase concentration. Literature on the enhancement and inhibition of the HAase activity is vast and contradictory in many cases (Krishnapillai et al., 1999) and these contradictions could be partly due to the existence of the dual role, activation and inhibition, of molecules, like proteins, which depends on the micro-environment. For example, does the optimal protein concentration depend on the microenvironment, and especially does it depend on the HA concentration, protein source, ionic strength, pH, etc? We present here a detailed study of the ternary HA / HAase / BSA system in order to answer a part of this question and to better understand how the competition between HAase and BSA to form complexes with HA can control the HAase activity. 2. Results 2.1. Influence of the BSA concentration on the hyaluronidase activity at pH 4 A series of experiments was performed at the unique 0.5 g L− 1 HAase concentration and at different HA concentrations ranging from 0.25 to 2 g L− 1. Fig. 1 represents the superimposition of the BSAdependencies of the initial HA hydrolysis rate, i.e. the initial HA

Fig. 1. Superimposition of the BSA-dependences of the HAase activity: Initial HA hydrolysis rates plotted against the BSA concentration at pH 4, for an HAase concentration of 0.5 g L− 1 and different HA concentrations: 0.25 g L− 1 (●), 0.50 g L− 1 (■), 1 g L− 1 (▲) and 2 g L− 1 (▼).

hydrolysis rate plotted against the BSA concentration, for the different HA concentrations. Fig. 1 shows that at constant HAase concentration, the optimal BSA concentration depended on the HA concentration. In the two cases corresponding to an HA concentration equal to or higher than 1 g L− 1, the shape of the curve was identical and showed three domains: In the first domain corresponding to BSA concentrations lower than 0.1 g L− 1, when the HA concentration was 1 g L− 1, the initial hydrolysis rate remained quite constant and very low. The BSA molecules formed complexes with HA but were not sufficient to entirely cover the HA molecules and prevent HAase binding on HA. HAase was thus entirely complexed with HA leading to a quasi zero reaction rate confirming that the complexed HAase is not catalytically active. In the second domain, corresponding to BSA concentrations ranging from 0.1 to 0.65 g L− 1 when the HA concentration was 1 g L− 1, the initial hydrolysis rate strongly increased up to the maximum because the concentration of the BSA molecules was high enough to prevent the binding of all the HAase molecules on HA. As the BSA concentration was increased, the free soluble HAase concentration increased, leading to a strong increase in the initial hydrolysis rate. This occurred because HA preferably formed complexes with BSA in comparison to HAase. For a BSA concentration of 0.65 g L− 1, almost the whole HAase was free in solution and the reaction rate was maximum. Finally, in the third domain, corresponding to BSA concentrations ranging from 0.65 to 4 g L− 1, when the HA concentration was 1 g L− 1, the initial hydrolysis rate decreased; HAase was entirely free in solution and not complexed with HA, but HA was more and more complexed with BSA. HAase had thus more and more difficulties to hydrolyse HA because of steric effects and/or hidden cleavable HA sites. In the two other cases corresponding to low HA concentrations, i.e. 0.5 g L− 1 and 0.25 g L− 1, the BSA-dependencies were slightly different. The first BSA domain did not exist because, even without any BSA, the HA concentration was not high enough to form complexes with the whole amount of HAase. The consequence was that, even without any BSA, there were some free HAase molecules in solution which were able to catalyse the HA hydrolysis, leading to a significant initial HA hydrolysis rate. The two other BSA domains, described above, existed and corresponded to the same phenomena as previously described. Finally, a fourth domain appeared for the highest BSA concentrations (BSA concentrations between 1 and 4 g L− 1 when the HA concentration was 0.25 g L− 1) where the initial hydrolysis rate remained close to

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zero. The reason was that the HA molecule was too tightly complexed with BSA to react with HAase. It means that all the cleavable HA sites were hidden by the BSA molecules. In fact, this fourth domain also existed for the two previous cases corresponding to high HA concentrations, but was not visible on the experimental curves because it should correspond to BSA concentrations higher than those experimentally tested. For all the cases, there was an optimal BSA concentration corresponding to the maximum in the initial HA hydrolysis rate and a critical BSA concentration above which the initial HA hydrolysis rate was nil. It is noteworthy that both the optimal and the critical BSA concentrations depended on the HA concentration. Depending on its concentration, BSA can thus be considered as an activator or an inhibitor of the HA hydrolysis catalysed by HAase.

2.2. The three noticeable points of the BSA — dependence of the HA hydrolysis catalysed by HAase A generalised BSA-dependence of the HA hydrolysis catalysed by HAase was thus drawn (Fig. 2) in which the four domains described above were delimited by three noticeable points. i) Point A, at the interface between domains ① and ②, corresponded to the concentration of the BSA molecules able to form additive complexes with the HA molecules already complexed with the whole HAase present in the system. This BSA concentration was noted [BSA]HAase zero because all the HAase molecules were complexed with HA and no free HAase molecules remained in solution leading to a quasi zero hydrolysis rate. ii) Point B, at the interface between domains ② and ③, corresponded to the minimum BSA concentration needed so that the HA molecules were complexed with BSA alone. In B, all the HAase molecules were free in solution and were able to catalyse the HA hydrolysis. The HA hydrolysis rate was maximum. This BSA concentration was noted [BSA]min. iii) Point C, at the interface between domains ③ and ④, corresponded to the highest BSA concentration able to produce a hydrolysable complexed HA. This BSA concentration was noted [BSA]max. At this point, the HA molecules were too tightly complexed with BSA to be accessible to HAase and the HA hydrolysis rate was nil. All the potentially cleavable HA sites were hidden by the BSA molecules.

Fig. 3. The three noticeable points in the BSA-dependence of the HAase activity corresponding to the limits for the stoichiometry of the electrostatic HA–BSA complexes. The numbers 1–4 correspond to the domains described in Fig. 2.

2.3. The four domains of the BSA — dependence of the HA hydrolysis catalysed by HAase Fig. 3 represents the values of [BSA]HAase zero, [BSA]min and [BSA]max plotted as a function of the HA concentration. The fact that the points corresponding to [BSA]min and [BSA]max were located on a straight line signified that there were constant ratios between [BSA]min and the HA concentration (ratio ψmin), and between [BSA]max and the HA concentration (ratio ψmax). These two ratios were given by the slope of the straight lines, 4.5 for ψmax and 0.65 for ψmin. These ratios were the limit ratios for the existence of pure hydrolysable HA–BSA complexes, since HAase was no longer complexed to HA. When the BSA over HA ratio was lower than ψmin, there was still some HAase molecules complexed with HA. When the BSA over HA ratio was higher than or equal to ψmax, there was no place on the HA molecule for supplementary BSA molecules, nor for the HAase molecules. The HA molecule was too tightly complexed with BSA to allow its enzymatic hydrolysis by HAase. When the BSA over HA ratio was ranged from ψmin to ψmax, the HA molecule was complexed with BSA in various complexion states. The BSA molecules were not too tightly complexed on the HA molecule and HAase could catalytically link the HA molecule and hydrolyse it. Statistically, the concentration of the hindered cleavable HA sites was proportional to the complexed BSA concentration, i.e. the BSA concentration in the reaction medium. This justified that the initial hydrolysis rate, which was proportional to the concentration of the accessible cleavable HA sites, linearly decreased when the BSA concentration was increased between B and C points. 2.4. The stoichiometry of the HA–BSA complexes

Fig. 2. Generalised BSA-dependence of the HAase activity with the four domains separated by the three noticeable points. Domain ① with no influence of BSA, domain ② with increase in the HAase activity by BSA, domain ③ with decrease in the HAase activity by BSA, and domain ④ with no influence of BSA.

In domain ③, it can be assumed that all the HA molecules were complexed with BSA and that all the HAase molecules were free in solution and catalytically active. We thus can consider the enzymatic system as a free enzyme with a binary HA–BSA complexed substrate. The binding of BSA on HA was minimal in B and maximal in C. By considering the molar masses of BSA (M = 69 000 g mol− 1) and HA (Mn = 0.976 · 106 g mol− 1) and the two ratios ψmin = 0.65 in B and ψmax = 4.5 in C, we can calculate that there were 64 molecules of BSA per HA molecule when the HA was tightly complexed with BSA (point C) and 9 molecules of BSA per HA molecule when HA is in its minimal complexion state with BSA (point B). It also means that 265 HA disaccharides were associated with one BSA molecule in B and 38 HA disaccharides were associated with one BSA molecule in C. Whatever

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the HA concentration, B points, as C points, characterised the HA–BSA complexes with a constant BSA over HA molar ratio, i.e. a constant stoichiometry. In the presence of HAase, the stoichiometry of the HA– BSA complexes was ranged from 9 and 64 BSA molecules per HA molecule. 2.5. The stoichiometry of the HA–HAase complexes When the HA concentration was equal to 1 g L− 1 (Fig. 1), point A corresponded to a BSA concentration of 0.1 g L− 1. It means that, in A, HA was entirely complexed with both HAase at 0.5 g L− 1 and BSA at 0.1 g L− 1. As mentioned in the previous paragraph, point B indicated that HA at 1 g L− 1 was entirely complexed with BSA when the BSA concentration was 0.65 g L− 1. It means that BSA at 0.1 g L− 1 occupied 15% of the HA molecule and thus that HAase at 0.5 g L− 1 occupied the remaining 85% of the HA molecule. It can thus be calculated that binding of 100% of the HA would require 0.60 g L− 1 of HAase. By taking into account the molar masses of HA and HAase (M = 57 000 g mol− 1), it can be deduced that a maximum of 10 molecules of HAase can be complexed per HA molecule. It also means that 237 HA disaccharides were associated with one HAase molecule. For a ternary HA / HAase / BSA system (domains ① and ②), the conclusion was that the binding of HAase on HA approximately corresponded to the same quantity of protein than the binding of BSA on HA. 2.6. Substrate-dependence of the HA hydrolysis catalysed by HAase in the presence of BSA The data of Fig. 1 can also be used to plot the substrate-dependence of the HA hydrolysis reaction for different BSA concentrations (Fig. 4). When there was a high BSA over HA ratio in the reaction medium, HA preferably formed complexes with BSA whilst HAase remained free in solution. At constant BSA concentration, the smaller the HA concentration, the tighter the BSA binding on the HA molecule. In that case, the substrate quality was not good and the initial hydrolysis rate remained very low, near zero. When the HA concentration was increased, the BSA molecules distributed on more and more HA molecules and these HA molecules were better substrates for HAase, resulting in an increase in the hydrolysis rate. Of course, as usually observed in enzymology, the initial hydrolysis rate also increased because of the increase in the substrate concentration. The increase in

Fig. 5. Substrate-dependence of the HAase activity when the substrate considered was HA complexed with BSA in the ψmin ratio: Initial HA hydrolysis rates plotted against the HA concentration at pH 4, for an HAase concentration of 0.5 g L− 1. Michaelis–Menten type behaviour of the HA / HAase/BSA system when HA complexed with BSA in the ψmin ratio (B point) is considered as substrate.

the HA concentration thus caused the increase in the initial hydrolysis rate by two additive ways: the increase in the substrate concentration and the increase in the quality of the substrate due to the decrease in the BSA density on the HA molecule. This constituted an apparent auto-catalytic mechanism and led to a sigmoidal curve (Fig. 4). At high HA concentrations, there was not enough BSA to form all the possible complexes with the HA molecules and some binding sites were accessible for HAase molecules, resulting in a decrease in the free HAase concentration and thus a decrease in the initial hydrolysis rate. In the presence of BSA, the substrate-dependence was thus atypical in the sense that the initial reaction rate showed a sigmoidal curve with a very low increase in the initial rate at low substrate concentrations, a maximum for an optimal substrate concentration and a decrease at high substrate concentrations. Even in the presence of BSA, when HA was considered as substrate, the substrate-dependence of HAase showed some similarities with a classical substrate-dependence of an enzyme with a mechanism of inhibition by excess of substrate. 2.7. Michaelis–Menten behaviour of the ternary HA / HAase / BSA system for constant complexion state of the substrate

Fig. 4. Superimposition of the substrate-dependences of the HAase activity: Initial HA hydrolysis rates plotted against the HA concentration at pH 4, for an HAase concentration of 0.5 g L− 1 and different BSA concentrations: 0.10 g L− 1 (●), 0.25 g L− 1 (■), 0.50 g L− 1 (▲), 1 g L− 1 (▼), 3 g L− 1 (♦) and 4 g L− 1 (○).

Point B represents the state of the system for which all the HAase molecules were free in solution and all the HA molecules were complexed with BSA in the most extended mode. In the case presented in Fig. 1, corresponding to the four BSA-dependences, the four B points were characterised by the same HAase concentration because all the HAase molecules were free in solution at that point, and by different concentrations of HA complexed with BSA in the same complexion state (ψmin = 0.65). It was thus possible to obtain the substrate-dependence of HAase with respect to the complexed HA as substrate, by plotting the values of the initial hydrolysis rates obtained at the different B points as a function of the HA (complexed with BSA) concentration at these B points. Fig. 5 shows that HAase behaved as a Michaelis–Menten type enzyme with respect to HA, complexed with BSA in the unique ψmin ratio. It has already been shown that the cleavable HA site must be considered as the substrate entity when kinetics studies were performed with HA / HAase systems (Vincent et al., 2003). In the present HA / HAase / BSA systems, the cleavable HA site must also be considered as the substrate entity, but at constant BSA concentration, the cleavable HA site concentration is no

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longer proportional to the HA concentration. The only situation where the cleavable HA site concentration is proportional to the HA concentration is encountered when the BSA over HA ratio is constant. B points corresponded to this situation and the substrate-dependence corresponding to the B points was thus of the Michaelis–Menten type. The Michaelis constants Vm and Km were thus determined by using the Lineweaver–Burk linearisation (Fig. 6) obtained by plotting the inverse of the initial hydrolysis rate as a function of the inverse of the substrate concentration; they were equal to 66 µmol L− 1 min− 1 for Vm and 0.28 g L− 1 for Km for a ψ ratio of 0.65. In fact, the ratio ψ defines the way by which the HA molecule is complexed by BSA. It corresponds to a given percentage of hydrolysable sites and of hidden sites. For a given ψ ratio, this percentage does not vary, whatever the HA concentration, and it defines the quality of the substrate molecule. Fig. 7 shows that a similar Michaelis–Menten type behaviour was observed in the substrate-dependences plotted for ψ ratios of 1, 1.5 or 2 corresponding to different complexion states for the HA molecule. It means that HAase followed a Michaelis–Menten type behaviour with respect to BSA-complexed HA when the BSA concentration was high enough to avoid the binding of HAase on HA (domain ③ between B and C points) and when HA was in a given complexion state. Finally, it was also possible to define a virtual point for which the HA molecule would not be complexed with BSA; this point was obtained by linearly extrapolating the domain ③ at zero BSA concentration. When the virtual reaction rates obtained for these points were plotted against the HA concentration, they were also in agreement with the Michaelis–Menten type behaviour of HAase (Fig. 7). 2.8. The HA–BSA complexes at pH 5.25 Similar experiments were performed at pH 5.25 with an HA concentration of 0.25 g L− 1, and BSA concentrations ranging from 0 to 10 g L− 1. The initial HA hydrolysis rates were determined as above and plotted against the BSA concentration (Fig. 8). The figure shows that the behaviour of the ternary HA / HAase / BSA system at pH 5.25 was the same than at pH 4. There was an optimal BSA concentration corresponding to a maximum in the initial HA hydrolysis rate (point B) and a critical BSA concentration above which the initial HA hydrolysis rate was nil (point C). Depending on its concentration, BSA can thus be considered as an activator or an inhibitor of the HA hydrolysis catalysed by HAase at pH 5.25. However, the effect of BSA was much lower at pH 5.25 than at pH 4 since much more BSA was needed to reach the maximum of the

Fig. 6. Lineweaver and Burk linearisation for the Michaelis–Menten type behaviour of the HA / HAase / BSA system when HA complexed with BSA in the ψmin ratio (B point) is considered as substrate.

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Fig. 7. Substrate-dependences of the HAase activity when the substrate considered was HA complexed with BSA in constant ratios: Initial HA hydrolysis rates plotted against the HA concentration at pH 4, for an HAase concentration of 0.5 g L− 1. Michaelis– Menten type behaviour of the HA / HAase / BSA system when HA complexed with BSA in constant ratios is considered as substrate. Ratios were equal to: 0 (♦) (virtual point), 0.65 (●), 1 (■), 1.5 (▲) and 2 (▼).

hydrolysis rate, i.e. to prevent the binding of the HAase molecules on HA (point B). For example, when the HA concentration was 0.25 g L− 1, [BSA]min was equal to 4 g L− 1 at pH 5.25 instead of 0.15 g L− 1 at pH 4. ψmin was thus equal to 16 at pH 5.25 instead of 0.65 at pH 4. The BSA concentration necessary to prevent the HAase binding was thus 25 times higher at pH 5.25 than at pH 4. The reason is that BSA was much less positively charged at 5.25 than at pH 4 because the value of its isoelectrical pH (pI) is close to 5.2 (Wang et al., 1996; Xu et al., 2000). Fig. 8 also shows that HA hydrolysis rates were much lower at pH 5.25 than at pH 4, including the case corresponding to point B. At this B point, all the HAase molecules were free in solution and the HAase concentration was the same (0.5 g L− 1) as it was at pH 4 (Fig. 1); it means that the HAase activity was much higher at pH 4 than at pH 5.25. However, the substrate was likely not the same at pH 4 than at pH 5.25, since HA was surrounded by much more BSA molecules at pH

Fig. 8. BSA-dependence of the HAase activity at pH 5.25: Initial HA hydrolysis rate plotted against the BSA concentration at pH 5.25, for an HAase concentration of 0.5 g L− 1 and an HA concentration of 0.25 g L− 1.

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5.25 than it was at pH 4. However, the present experiments clearly show that, at pH 5.25, electrostatic complexes were still formed between HA and BSA and were able to prevent the HA–HAase complex formation. This was likely related to the presence of positive patches on the surface of the BSA molecule (Mattison et al., 1998; Park et al., 1992). 3. Discussion and conclusion It has been shown, at pH 4 and under low ionic strength conditions, that BSA is able to compete with HAase to form electrostatic and noncatalytic complexes with HA. The presence of BSA in an HA–HAase reactive medium prevents the binding of HAase which remains free and retains its catalytic activity. However, the influence of the BSA concentration on the HAase activity shows a maximum in the HAase activity which corresponds to the optimum BSA concentration necessary to keep all the HAase molecules free in solution. At higher BSA concentrations the HAase activity decreases. At lower BSA concentrations the HAase activity increases when the BSA concentration is increased. Several experiments performed with different HA concentrations and with the same HAase concentration have shown that i) the shape of the BSA-dependencies remains identical, ii) the optimum BSA concentration is proportional to the HA concentration, and iii) the maximum reaction rate increases when the HA concentration is increased. The general scheme for the BSA-dependence is thus characterised by four domains: a first domain where addition of BSA does not affect the hydrolysis rate, a second domain where addition of BSA increases the hydrolysis rate up to a maximum, a third domain where addition of BSA decreases the hydrolysis rate and a fourth domain at high BSA concentrations where the hydrolysis rate remains nil. These domains are delimited by three noticeable points corresponding to constant BSA over HA mass ratios ψ equal to 0.1, 0.65 and 4.5, respectively. This confirms the notion of HA–protein complexes we have proposed to explain the atypical kinetic behaviour of the HA / HAase system (Lenormand et al., 2007, 2008; Deschrevel et al., 2008). The analysis of the BSA-dependence curve has allowed us to determine the stoichiometries of both the HA–BSA and the HA–HAase complexes, close to 10 molecules of protein per HA molecule when the molar mass of the HA molecule is close to 1 MDa. This stoichiometry represents the maximum quantity of protein, either BSA or HAase, able to form electrostatic complexes when they are co-complexed with HA, i.e. when the complex formation is ternary. When HAase is no longer complexed with HA, the binding of BSA on the HA molecule becomes binary and can increase up to 64 BSA molecules per HA molecule. Concerning HAase, the stoichiometry of 10 molecules of protein per HA molecule is in good agreement with previous experiments performed at pH 5 (Astériou, 2002) since the HAase-dependence suggests that an HA solution at 1 g L− 1 is able to form non-reactive complexes with 0.7 g L− 1 of HAase. We have also shown that HAase recovers a Michaelis–Menten type behaviour when we consider for substrate the HA molecule complexed with BSA in the same complexion state, i.e. with the same BSA over HA ratio ψ. Finally, we have shown that the same behaviour is observed at pH 5.25 with some quantitative differences, especially a lower hydrolysis rate and a lower efficiency of BSA because pH is close to the pI of BSA. The fact that there is no hydrolysis reaction when HA at 1 g L− 1 and HAase at 0.5 g L− 1 were mixed together shows that i) all the HAase molecules form complexes with HA, and ii) the HAase molecule complexed to HA is no longer catalytically active. Consequently, the dissociation constant for the electrostatic HA–HAase complex is very low. In a previous paper (Lenormand et al., 2008), we have shown that i) all the HA molecules were complexed with BSA when HA at 0.2 g L− 1 and BSA were mixed together at a concentration ratio higher than ψmax, and ii) all the BSA molecules were complexed with HA when HA at 0.2 g L− 1 and BSA were mixed together at a concentration ratio lower than ψmax. This signifies that the dissociation constant for the electrostatic HA–BSA

complex is also very low. The dissociation constant for the HA-lysozyme complex has been estimated to 10− 7 M, higher than the constant for the specific complex HA-HN (Maingonnat et al., 1999; Van Damme et al., 1994). Although it is very difficult to compare the values of the equilibrium constants, the facts that i) BSA was able to prevent the binding of HAase on HA, and ii) the addition of BSA in an HA / HAase system leads to an increase in the hydrolysis rate correlated to the release of HAase molecules (Lenormand et al., 2008), show that the dissociation constant for the electrostatic HA–BSA complex is much lower than that for the electrostatic HA–HAase complex. The dissociation constant for the electrostatic HA–HAase complex is thus much higher than 10− 9 M. This supports the fact that all the HAase molecules are free in solution at the optimal BSA concentration (point B). BSA can thus be considered as both an activator for the HA / HAase system when considered at low concentration by avoiding the binding of HAase on the HA molecule and an inhibitor when considered at high concentration by covering the HA molecule. In fact, the enhancement of the HAase activity in the presence of BSA, is not an activation of the enzyme by itself, but rather the suppression of its inhibition due to the abolishment of the complex formation of the enzyme molecules with HA. Because of the electrostatic nature of the HA–protein interactions, this occurs mainly at low levels of ionic strength, but recent experiments have shown that the phenomenon of complex formation still exists at 0.15 M ionic strength, which is usually considered as the physiological ionic strength, and at low HAase over HA ratios (Lenormand et al., 2007, 2008). The apparent activation/inhibition of HAase by proteins, such as BSA, has been reported several times in the literature without any definitive explanation. These results have very important implications on the measure of the HAase activity in biological fluids, and especially in serum or plasma, since it has been shown that the HAase activity measured in serum could be a signal of liver damages (George and Stern, 2004) and the HAase activity measured in plasma a tumour indicator (Laudat et al., 2000). This explains why several research groups are nowadays involved in measuring the exact value of the HAase activity in biological fluids. Our experiments have pointed out the influence of the presence of non-catalytic proteins on the apparent HAase activity in such fluids and our results are of great importance for measuring the actual HAase activity, especially when the HAase concentration is low. In point B, for which the protein concentration is optimal, all the HAase molecules are free in solution and the hydrolysis rate obtained under these conditions is thus well correlated to the true HAase concentration. However, the hydrolysis rates and thus the Vm value concern a substrate different from pure HA, since in B, HA is complexed by the added protein. Vm values thus depend on the nature of the added protein. The actual HAase activity is a combination of the intrinsic enzyme activity and the free enzyme concentration which depends on complex formation. Experiments performed at pH 5.25 show that modulation of the HAase activity by BSA greatly depends on the pI value of BSA. It means that replacing BSA by another protein with a higher pI value, such as lysozyme, allows activation and/or inhibition of the HAase activity at higher pH values including pH 7 (submitted paper). It shows that testicular HAase may be active at neutral pH, depending on the micro-environment, although it is considered as an acid-active enzyme, like most of the HAases. The actual pHdependence and the actual optimal pH are also dependent on the micro-environment and especially on the proteins or polyelectrolytes present in the medium. The atypical behaviour of the initial reaction rate of the HA hydrolysis as a function of the HA concentration under low ionic strength conditions is characterised by a highly non-linear and non-monotonous relationship. Such behaviour could be of great importance in extracellular matrix regulation, under either normal or pathological conditions. On the one hand, although tumoural HAase (Delpech et al., 1987) is different from testicular HAase, there are many similarities between

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the two enzymes, particularly concerning the kinetic behaviour (Astériou, 2002). Atypical behaviour such as that ascribed with BTHAase could be of great interest in cancer research since many authors have already mentioned that there are high HA and HAase concentrations in tumour tissues compared to normal tissues (Bertrand et al., 1997; Victor et al., 1997; Knudson et al., 1989; Manley and Warren, 1987; Cooper and Forbes, 1988; Lokeshwar et al., 1997; Das and Chatterjee, 1981; FiszerSzafarz and Gullino, 1970; Fiszer-Szafarz and Szafarz, 1973). In fact, the behaviour of BTHAase and that of tumour-associated HAase are similar (Astériou, 2002). These conclusions are also very important because of cancer for which the problem of inhibition of HAase is nowadays crucial (Mio and Stern, 2002). The inhibitors of the HAase activity are still uncharacterised and many studies were undertaken in the last decade to understand the inhibition phenomenon and define the inhibitor. The actual tendency is to consider that HAase inhibitors are ubiquitous in mammalian tissues and biological fluids such as plasma, serum, urine and saliva. Most of the studies have concerned serum. It has been suggested that the predominant serum inhibitor is magnesiumdependent and is eliminated by protease or chondroïtinase digestion (Mio et al., 2000). Additional studies suggest that the circulating inhibitor has the characteristics of the inter-α-inhibitor (IαI) family (Mio et al., 2000) and that a covalent linkage between the IαI and polysaccharides, such as chondroïtin sulfate, is required for HAase inhibitory activity (Mio et al., 2000). HA linked to IαI also inhibits HAase, but no answer is reported concerning the significance of this association and its role in the HAase inhibition. Other studies have reported that lysosomal HAase is inhibited by sulfated polysaccharides, most probably through electrostatic interactions (Aronson and Davidson, 1967). Heparin and mucin have also been mentioned as inhibitors for testicular HAase (Meyer et al., 1960). All these reported facts and suggestions underline the importance of polysaccharides and polysaccharide– protein associations in the HAase inhibition phenomenon. Another very interesting suggestion is that the regulation and homeostasis of the ECM could be better realized by HAase-associated catabolism of HA and its modulation by inhibition, than by stimulation of its synthesis by HA synthases (Mio and Stern, 2002). This point of view suggests that HAase would be present in every tissue containing HA, that is generally observed, but it also suggests that HAase would be retained in an inactive form, for example bound to inhibitors (Mio and Stern, 2002), to avoid great havoc in healthy tissues. Our studies suggest that high molecular weight HA could be a good candidate for this type of ubiquitous and non-specific inhibition which can be reversed by pulses of ionic strength or protein fluxes, and involved in their regulation in the ECM. 4. Experimental procedures 4.1. Materials Bovine testicular HAase (H 3884, lot 38H7026), BSA (A 3675, lot 78H1399) and Sodium hyaluronate from human umbilical cord (H 1876, lot 127H0482) were obtained from Sigma. The molar mass of HA was close to 1 MDa. More precisely, the number-average molar mass (Mn) of HA was 0.967 · 106 g mol− 1 and its polydispersity index, which represents its degree of homogeneity, was 1.45. The molar mass of BSA was 69 · 103 g mol− 1. HA, BSA and HAase were used without any further purification. The chemicals used in the assays were: sodium tetraborate (Prolabo 27 727–297), sulfuric acid (Sigma S 1526), carbazole (Sigma C 5132), boric acid (Sigma B 7660), p-dimethylaminobenzaldehyde (DMAB) (Sigma D 8904), glacial acetic acid (Sigma A 6283), N-acetylD-glucosamine (Sigma A 8625) and sodium glucuronate (Sigma G 8645). Absorbance was measured using a Uvikon 860 Kontron spectrophotometer equipped with a temperature-controlled chamber and connected to a computer. pH adjustments were carried out using a Metrohm 632 pH-meter equipped with a Radiometer Analytical XC161 electrode.

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4.2. Methods 4.2.1. Uronic acid assay The HA mother solution (weighed at 10 g L− 1) was prepared in milli-Q water and assayed by the method described by Dische (1947) and modified by Bitter and Muir (1962). The experimental procedure using carbazole was extensively described in our previous papers (Vincent et al., 2003; Astériou et al., 2001). Sodium glucuronate was used for calibration. 4.2.2. N-acetyl-D-glucosamine reducing end assay Measurement of the concentration of N-acetyl-D-glucosamine reducing ends was based on the method described by Reissig et al. (1955). Because of the presence of HAase in the samples, turbidity and colour were simultaneously present. We thus used the improvement of the Reissig method, described by Astériou et al. (2001). The experimental procedure using DMAB was extensively described in our previous papers (Vincent et al., 2003; Astériou et al., 2001). N-acetylD-glucosamine was used for calibration. The reducing end concentration defines the HA chain concentration. 4.2.3. Kinetics of the HA hydrolysis Adequate volumes of the HA and BSA mother solutions were placed in a reactor, diluted to the desired concentrations with milli-Q water, adjusted to the desired pH with HCl (or KOH), stirred and maintained at 37 °C. After 2 min, the reaction was started by adding an adequate volume of a concentrated HAase solution (10 g L− 1 in milli-Q water). At each time point, a 200 µL aliquot of the reaction mixture was removed from the reactor and assayed by using the N-acetyl-D-glucosamine reducing end assay. For each kinetics, the hydrolysis reaction was followed for 3 h and the concentration of the HA reducing ends was plotted against time. The method thus gave the time evolution of the HA chain concentration; the slope of the tangent to that curve directly represented the reaction rate. The kinetic curve was then fitted by the biexponential model developed by Vincent et al. (2003) and the initial reaction rate was calculated as being equal to the value of the first derivative of that function at time zero. Acknowledgements We thank Dilys Moscato and Vishwas Purohit for critical reading the manuscript. We are grateful to the “Matériaux Polymères, Plasturgie” network for the fellowship granted to Frédéric Tranchepain and to the French Research and Technology Ministry for the fellowship granted to Hélène Lenormand. References Aronson Jr., N.N., Davidson, E.A., 1967. Lysosomal hyaluronidase from rat liver: II. Properties. J. Biol. Chem. 242, 441–444. Astériou, T., 2002. Dr. Sci. Thesis, Rouen, France. Astériou, T., Deschrevel, B., Delpech, B., Bertrand, P., Bultelle, F., Merai, C., Vincent, J.C., 2001. An improved assay for the N-acetyl-D-glucosamine reducing ends of polysaccharides in the presence of proteins. Anal. Biochem. 293, 53–59. Astériou, T., Gouley, F., Deschrevel, B., Vincent, J.C., 2002. Influence of substrate and enzyme concentrations on hyaluronan hydrolysis kinetics catalyzed by hyaluronidase. In: Kennedy, J.F., Phillips, G.O., Williams, P.A. (Eds.), Hyaluronan, vol. 1. Woodhead Publishing, Wrexham, Wales, pp. 249–252. Astériou, T., Vincent, J.C., Tranchepain, F., Deschrevel, B., 2006. Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength. Matrix Biol. 25, 166–174. Bertrand, P., Girard, N., Duval, C., d'Anjou, J., Chauzy, C., Menard, J.F., Delpech, B., 1997. Increased hyaluronidase levels in breast tumor metastases. Int. J. Cancer 73, 327–331. Bitter, T., Muir, H.M., 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4, 330–334. Cameron, E., 1966. Hyaluronidase and Cancer. Pergamon, Oxford. Catterall, J.B., 1995. Hyaluronic acid, cell adhesion and metastasis. Cancer J. 8, 320–324. Cooper, E.H., Forbes, M.A., 1988. Serum hyaluronic acid levels in cancer. Brit. J. Cancer 58, 668–669.

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