Disc gel electrophoresis of the vegatative cell -free extracts ofstrains of Clostridium botulinum types A
Disc gel electrophoresis of the vegatative cell -free extracts ofstrains of Clostridium botulinum types A, B, C, E, and F and therelated nontoxic group showed limited value as a means for identifyingthese closely related microorganisms, since separation,though not consistent in all cases, could only be based on the numberof protein fractions in the gel,Enzyme staining of the protein -laden polyacrylamide gels ofthe strains showed single or multiple molecular forms for malic(NAD and NADP), isocitric (NAD), succinic (NAD), and lactic (NAD)dehydrogenases and alkaline phosphatase. Analyzing the enzymepatterns of the strains revealed that most of these enzyme systemsare useful for distinguishing the types and the nontoxic strains.A method which allowed two samples to he run in the samepolyacrylamide gel showed that the differences between the total1 protein patterns of two strains can be demonstrated clearly.A type of iron bound protein (ferredoxin) was isolated from C.botulinum using a modification of the method recommended by L. E.Mortenson for isolating ferredoxin from Clostridium pasteurianum.The protein exhibited maximum absorption in the ultraviolet regionnear 260 mμ. Portions of the isolated iron bound protein wereseparated by disc electrophoresis, and following specific iron boundprotein staining, showed a positive reaction in the same position inthe gel column as first demonstrated using cell -free extract.Evidence accumulated using cell -free extract of C. botulinumsuggests that pyruvate is metabolized through a phosphoroclasticsystem as demonstrated in other clostridia. It is probable that theferredoxin has the important role of electron mediator betweenpyruvic oxidase and hydrogenas a for hydrogen evolution and acetylphosphate formation.A purposed system for the synthesis of aspartate and glutamatein C. botulinum incorporating the above enzymes including those ofthe phosphoroclastic system in a partial citric acid cycle andglyoxylate bypass was described.