Please use this identifier to cite or link to this item:
Title: Studies on the hyaluronidase enzyme purified from the venom of Chinese red scorpion Buthus martensi Karsch
Authors: FENG LUO
Keywords: Scorpion venom, hyaluronidase, purification, RACE PCR, full protein sequence, CD44
Issue Date: 19-Aug-2009
Citation: FENG LUO (2009-08-19). Studies on the hyaluronidase enzyme purified from the venom of Chinese red scorpion Buthus martensi Karsch. ScholarBank@NUS Repository.
Abstract: The present work includes 1) screening of the biological activities in scorpion Buthus martensi Karsch (BmK) crude venom; 2) the purification and characterization of the hyaluronidase enzyme (BmHYA1) from the venom of BmK; 3) the cDNA cloning and expression of BmHYA1 and 4) the preliminary pharmacological study of BmHYA1. Scorpion venom is a rich source for short neurotoxic peptides but this study indicates it also contains various high molecular weight (M.W.) proteins. A number of enzymatic activities have been detected in the present work including L-amino acid oxidase (LAAO), serine protease, and hyaluronidase. It is also possible to contain Phospholipase A2 (PLA2) and metalloproteinase. This work should be the pioneer in comprehensive investigation of the enzymatic proteins in scorpion BmK venom. The hyaluoridase from the crude venom of BmK, later named as BmHAY1, was studied in detail. The enzyme was purified from the crude venom by a successive chromatography of gel filtration, ion-exchange and reversed-phase high-performance liquid chromatography (RP-HPLC). The homogeneity was manifested by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and Edman degradation. MALDI-TOF result also showed its molecular weight of 48,696 Da. Its N-terminal amino acids were determined by Edman degradation and showed homologies to other venom hyaluronidases to some degree. BmHYA1 has an optimal temperature of 50 oC and optimal pH of 4.5. Its Km and Vmax are determined to be 95.3 µg/mL and 3.9 µg/min, respectively. Additionally, the enzyme can hydrolyze the substrate hyaluronan into tetrasacchrides. Rapid amplification of cDNA ends- polymerase chain reaction (RACE PCR) technique was used to clone the 3¿ end BmHYA1 cDNA sequence. The 5¿ degenerate primer was designed based on known N-terminal sequence hence the whole mature BmHYA1 sequence was deduced. Thisis also the first hyaluronidase full protein sequence from the scorpion species. The alignment shows it has some homologies (up to 34%) to other Glycol-Hydro-56 family members. The phylogenetic analysis indicates early divergence and independent evolution of BmHYA1 from other hyaluronidase family members. The recombinant BmHYA1 was expressed in E.coli but did not show the activity. The treatment with BmHYA1 to MDA-MB-231 breast cancer cells gave rise to the removal of hyaluronan from the cell surfaces. The further study about its effect on CD44 molecules showed that the environmental hyaluronidase (BmHYA1) can modulate the expression of CD44 variant 6.
Appears in Collections:Ph.D Theses (Open)

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
FengL.pdf6.75 MBAdobe PDF



Page view(s)

checked on Apr 18, 2019


checked on Apr 18, 2019

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.