Purification, characterization and molecular cloning of Vibrio fluvialis hemolysin.
Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.
Han
Jeong-Hyun
JH
Department of Biotechnology and Bioengineering, Pukyong National University, Pusan, 608-737, South Korea.
Lee
Jong-Hee
JH
Choi
Yoon-Hyeok
YH
Park
Je-Hyeon
JH
Choi
Tae-Jin
TJ
Kong
In-Soo
IS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D002413:Cations, Divalent; D002645:Chickens; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000378:metabolism; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006461:Hemolysis; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009995:Osmosis; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D006460:Hemolysin Proteins; C073845:hemolysin, Vibrio