Isolation, Characterization And Mechanism Of Hyaluronate Degrdation By Hyaluronidase From Yak(Bos Grunniens) Testis

Hyaluronidase has been used to catalyze the hyaluronan found in the extracellular matrix. The major final reaction products interact with various receptors of the extracellular matrix, which plays an important role in cell invasion and anti-angiogenesis. However, these hyaluronidases purified by various methods are a complex mixture of proteins. Our aim was to develop an effective isolation process based on chromatography to obtain a novel hyaluronidase with higher activity and purity from Bos grunniens testes. The enzymatic characterization, and the mechanism for hyaluronan degradation investigated here may contribute to the optimization of therapies for clinical application and help in understanding the numerous pathophysiological processes in which the enzyme is involved. The results showed that:1. The optimum extraction parameters of Yak testis hyaluronidase was confirmed. The best extraction condition of Yak testis hyaluronidase was as follows: 50 m M sodium acetate buffer(p H 4.98) as extractant, ratio of material to liquid 1:4, extraction time 33 h. In order to reduce the loss of enzyme activity. It was necessary to add Na Cl(150 m M), BSA(150 m M), and SAL(2.28 m M) in the buffer. The enzyme activity, specific activity, and yield of crude enzyme was 278 U, 1.31 U/mg, 1.06%, respectively.2. The optimum purification parameters of Yak testis hyaluronidase was confirmed. Crude enzyme was isolated by cation exchange chromatogram(SP Sepharose Fast Flow). The elution buffer was 50 m M sodium acetate buffer, p H 6.0, containing 1.0 M Na Cl. The flow rate was 5 m L/min. Step elution was performed by sequential addition of the same buffer at increasing ionic strengths from 0.05 to 0.5 M(0.05 M、0.1 M、0.15 M、0.2 M、0.3 M、0.4 M、0.5 M Na Cl). Afterwards, Superdex 75 PG was used to finish final purification of Yak testis hyaluronidase. The elution buffer was 50 m M sodium acetate, containing 150 m M Na Cl, p H 6.0, at a flow rate of 0.5 m L/min.3. Obtained a high purity Yak testis hyaluronidase. Crude enzyme was purified through SP Sepharose Fast Flow and Superdex 75 PG. The purity was confirmed by a reverse phase FPLC Shodex C4 column. The specific activity, purification fold, and yield of Yak testis hyaluronidase in the first step was 11.22 U/mg, 8.56, 65.76%, respectively. The specific activity, purification fold, and yield of Yak testis hyaluronidase in the final step was 42.27 U/mg, 32.25, 15.57%, respectively. It was a monomeric protein of 55 k Da.4. The enzymatic characteristics of Yak testis hyaluronidase was confirmed. Yak testis hyaluronidase exhibited maximum activity at 37°C, and a specificity to sodium hyaluronate higher than that of chondroitin-4-sulphate, chondroitin-6-sulphate, and dermatan. The Km value was 0.102 mg/m L. Activity of Bg Hya1 was inhibited significantly by Fe3+, Mg2+, and heparin. In terms of storage stability, 92% of the activity was retained after four weeks at 4°C. The optimum p H of Bg Hya1 was 3.8 and 5.8 by using different method.5. HA degradation at acidic p H producing even oligosaccharides of one kind only. HA degradation at p H 3.8 for 3 h, yielded a series of even oligosaccharides with NAG at the reducing end. HA tetrasaccharide(776.5 Da), hexasaccharide(1155.6 Da), octasaccharide(1534.7 Da), decasaccharide(1913.8 Da), and dodesaccharide(2293.4 Da) were detected as the predominant products. After incubation for 24 h, the concentration of HA tetrasaccharide and hexasaccharide increased, and decreased the amounts of HA octasaccharide, decasaccharide, and dodesaccharide.6. HA degradation at weakly acidic p H producing odd and even oligosaccharides. HA degradation at p H 5.8 yielded five kinds of even oligosaccharides with NAG at the reducing end and three kinds of odd oligosaccharides with Glc UA at the reducing end. Longer incubation periods increased the amounts of trisaccharide(573.4), pentasaccharide(952.5 Da), and heptasccharide(1331.6 Da).7. HA even oligosaccharide was degraded by Yak testis hyaluronidase. As the substrate, HA and HA even oligosaccharides were degraded by Yak testis hyaluronidase at p H 3.8. HA Octasaccharide was the minimum substrate for Yak testis hyaluronidase. It explained why the concentration of HA tetrasaccharide and hexasaccharide increased after degradation for 24 h.8. Hyaluronan was degraded by Yak testicular hyaluronidase in two pathways. HA degradation at p H 3.8 yielded a series of even oligosaccharides with NAG at the reducing end. Meanwhile, as the substrate, even oligosaccharides were degraded by Yak testis hyaluronidase, which yielded amount of HA tetrasaccharide and HA hexasaccharide. The minimum substrate was HA octasaccharide. Longer incubation periods increased the amounts of HA tetrasaccharide and HA hexasaccharide. After incubation for 24 h, HA tetrasaccharide and HA hexasaccharide were detected as the predominant products. The other pathway was : HA degradation at p H 5.8 yielded a series of even oligosaccharides with NAG at the reducing end and odd oligosaccharides with Glc UA at the reducing end. Longer incubation periods decreased the amounts of odd oligosaccharides with a higher molecular weight. The formation of odd oligosaccharides was not caused by stop the enzyme reaction and the even oligosaccharide degradation.