RESUMO
Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis. The molecular weights, estimated by gel filtration, were 8400 and 19,000; by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, the values were calculated as 15,500 and 27,000. The electrophoretic bands were best detected by the periodic acid--Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19,000 hemolysins. However, trypsin treatment cleaved the 19,000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19,000 hemolysin and the smaller hemolysin was absent.
Assuntos
Proteínas Hemolisinas/análise , Klebsiella pneumoniae/análise , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Proteínas Hemolisinas/isolamento & purificação , Hemólise , Peso Molecular , Tripsina/farmacologiaAssuntos
Meios de Cultura/farmacologia , Pigmentos Biológicos/biossíntese , Infecções por Pseudomonas/microbiologia , Pseudomonas/classificação , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , Diagnóstico Diferencial , Humanos , Pseudomonas/metabolismo , Infecções por Pseudomonas/diagnóstico , Especificidade da EspécieRESUMO
An investigation of Klebsiella pneumoniae hemolytic activity was carried out. Strains isolated from different infected specimens were hemolytic in tryptic soy agar with rabbit blood; incubation at 4 degrees C enhanced the hemolysis. There was no evident red blood cell lysis in plates with human, sheep, mouse, and chicken erythrocytes. The culture in tryptic soy broth, its supernatant and bacterial lysate did produce evident hemolysis of rabbit red blood cells when they were preincubated with 2-mercaptoethanol. Klebsiella pneumoniae hemolysin showed the Arrhenius effect, while temperatures over 60 degrees C for 10 min reduced the activity of crude hemolysin to zero; purified hemolysin, however, was heat stable. Two hemolysins active on rabbit red blood cells were purified and both shared several properties. This work represents the initial description of a thiol-activated hemolysin in Gram-negative bacteria.
Assuntos
Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Klebsiella pneumoniae , Mercaptoetanol/farmacologia , Animais , Galinhas , Humanos , Técnicas In Vitro , Camundongos , Coelhos , OvinosRESUMO
The hemolytic activity of sixty K. pneumoniae strains was investigated in tryptic soy agar with rabbit, dog, sheep, human, chicken and mouse blood. All of them were lytic only for rabbit red cells. In liquid medium it was necessary a 2 mercaptoethanol treatment to detect a good degree of hemolysis. Cultures in tryptic soy broth gave 100% hemolysis in assays with rabbit erythrocytes and only when hemolysin was concentrated by purification was it active on dog and sheep but in a lesser degree (7.5% hemolysis). Supernatants of cultures were precipitated at different saline concentrations; the fraction obtained with 30-50% (NH4)2SO4 had hemolytic activity after dialysis and 2 mercaptoethanol treatment. Then this fraction was eluted in a Sephadex G-100 column, but electrophoresis in polyacrylamide gel showed that the hemolytic molecules obtained by gel filtration were contaminated with protein structures which had different electrophoretic migration. Ion-exchange chromatography showed best purification index and the recovery of activity was over 100%, it was possible to explain this good recovery once an inhibitor was detected. Two rabbit red cell lysins were purified, both shared several properties: SH-activation, pH optimum, thermolability, selectivity for rabbit red cells, mechanism of action, inhibition by cholesterol and divalent cations.
Assuntos
Proteínas Hemolisinas/isolamento & purificação , Klebsiella pneumoniae/análise , Animais , Galinhas , Cromatografia por Troca Iônica , Cães , Humanos , Camundongos , Oxigênio/farmacologia , Coelhos , Ovinos , Especificidade da EspécieRESUMO
The hemolytic activity of sixty K. pneumoniae strains was investigated in tryptic soy agar with rabbit, dog, sheep, human, chicken and mouse blood. All of them were lytic only for rabbit red cells. In liquid medium it was necessary a 2 mercaptoethanol treatment to detect a good degree of hemolysis. Cultures in tryptic soy broth gave 100
hemolysis in assays with rabbit erythrocytes and only when hemolysin was concentrated by purification was it active on dog and sheep but in a lesser degree (7.5
hemolysis). Supernatants of cultures were precipitated at different saline concentrations; the fraction obtained with 30-50
(NH4)2SO4 had hemolytic activity after dialysis and 2 mercaptoethanol treatment. Then this fraction was eluted in a Sephadex G-100 column, but electrophoresis in polyacrylamide gel showed that the hemolytic molecules obtained by gel filtration were contaminated with protein structures which had different electrophoretic migration. Ion-exchange chromatography showed best purification index and the recovery of activity was over 100
, it was possible to explain this good recovery once an inhibitor was detected. Two rabbit red cell lysins were purified, both shared several properties: SH-activation, pH optimum, thermolability, selectivity for rabbit red cells, mechanism of action, inhibition by cholesterol and divalent cations.
