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Title: Staphylococcus alpha toxin
Author: Arbuthnott, John Peebles
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1964
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The present thesis describes the general properties mode of action in vivo and in vitro, and a method of purification, of staphylococcal alpha toxin. The optimum pH and temperature of the haemolytic reaction were found to be 5.5 and 31 C. respectively. In proteolytic digestion and heat sensitivity, the toxin behaved as a typical protein. Contrary to current views alpha toxin was found intrinsically heat sensitive. A revised explanation of the paradoxical "Arrhenius phenomenon" is suggested and disscused in light of this. Alpha toxin was not used up in haemolysis; it acts as a catalyst. Kinetic experiments showed that at low concentrations of toxin the relationship between the rate of haemolysis and concentration was compatible with an enzymic reaction. At high concentrations there was a marked falling off; possible reasons for this are suggested and discussed , and the limitations or haemolysis as an indicator system are pointed out. Also the velocity of haemolysi was dependent of the concentration of rabbit red blood cells. Considering the talk of evidence it is concluded that alpha toxin belongs to that group of bacterial haemolysins which are thought to be enzymes. Divalent ions are not required for haemolysis. Inhibitors of bacterial protenses had no effect. Heavy metal ions such as Hg 2+ were found to inhibit at concentrations of 10-3M; below this they became haemolytic by themselves . Some organic sulphydryl inhibitors also inhibited alpha toxin; it seems therefore that free SH groups may play a role in haemolysis. The trypanocidal drug Suramin and related substances were powerful inhibitors of both the haemolytic and the lethal activity of alpha toxin. Phospholipids were tested as possible competitive inhibitors of alpha toxin in an attempt to gain information concerning the point of attack. Apart from a crude preparation of sheep brain cephalin none of them inhibited. The possible significance of this is discussed. Death from alpha toxin was found to be dose dependent: it was either very rapid, or occurred after considerable delay. Dose dependence and the pattern of death was largely the same for the four species examined, viz., rabbits, mice, fowl and frogs. Rapid death in seconds or minutes, without histological lesions is most likely explained by an action on heart or central nervous system. Slow death in several hours or days may result from lesions in a multiplicity of organs. Large doses( which killed rapidly intravenously) when administered subcutaneously or intraperitoneally killed much slower, probably because of the time required for diffusion of toxin to vital organs. Alpha toxin caused local flaccid paralysis of voluntary muscle when injected into the dorsal sac of 6 week old mice; at high doses this occurred before any detectable histological lesion. Muscles of the paralysed limbs not respond to electrical stimulation IN SITU. In presence of alpha toxin the reactivity of excised voluntary muscles of mice and frogs to acetyl choline and electrical stimulation was abolished IN VITRO. Muscles of curarised mice beghaved in the same way, and it concluded that alpha toxin has a direct myotoxic action. Heart muscle appeared less sensitive. Hearts of mice killed with alpha toxin continued to beat for a few minutes after death and hearts of frogs for up to several hours. In tissue culturos mouse heart explants were less affected than the whole animal. Whereas 128 MHD is an L.D. 50 for 20g. mice,explants of 20-1,000 cells were only moderately affected by 2,000 MHD/ml: some explants continued to beat, some stopped after 30 min., but recovered overnight. The possible significance of this is discussed. Alpha toxin, purified by combination of gel filtration on G.75 and DEAE gamma 50 Sephadex and fractional methanol precipitation, behaved as serologically and physically homogencous protein. The sedimentation constant was 3.1 S at 0,13% protein, which is close to that of 3.0 S suggested recently by Bernheimer and Schwartz (1963). The potency was 119,000 MH/mg. protein. The product was unstable and evidence was obtained which suggested that on standing or dialysis alpha toxin tends to polymerise with the appearance of heavy component.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available