VOL. 12 (1953) AMINO ACID INTERACTIONS IN STRICT ANAEROBES I05 



Rat brain homogenate 



Rats were killed by decapitation, the brains removed immediately and homogenized in i % 

 nicotinamide solution with a Potter-Elvehjem homogenizer. One g of brain was homogenized with 

 4 ml nicotinamide solution, 0.5 ml homogenate being used when required. 



Pigeon liver acetone powder 



Preparation. Pigeons were killed by decapitation and bled. The livers were removed and chilled. 

 After mincing, each liver was homogenized in the cold. The homogenized tissue was poured into 10 

 times its volume of acetone. The dehydrated material was filtered on a Buchner funnel and was 

 washed three times with acetone. The washed material was transferred to filter paper and dried in the 

 air for approximately 5 minutes. Drying was completed in a vacuum dessicator over calcium chloride. 



Extraction of liver acetone powder 



The powder was ground in the cold with water or 0.2 M KF, using a Potter-Elvehjem homogenizer. 

 After standing in ice for 10-15 minutes, it was centrifuged in the cold at 20,000 g for 2 minutes. To 

 «ach vessel was added 0.5 ml of the supernatant. 50 mg of the crude powder was used for each vessel. 



Estimation of the amino acids by paper chromatography 



0.02-0.04 nil solution, to be analysed, was spotted on Whatman No. i filter paper. After drying 

 at room temperature, the papers were chromatographed for iS hours in butanol-ethanol-ammonia- 

 water (20-60-7-13) by ascending chromatography. After drying, the papers were sprayed with 1% 

 ninhydrin to locate the amino acids. The areas containing the amino acids to be analysed were cut out 

 with a rim of 2 centimeters. The individual areas of filter paper were inserted into test-tubes, and 

 saturated with 5 % ninhydrin in butanol-water. Maximum colour was developed by incubation for 5 

 minutes at room temperature followed by 5 min at 55-60° C and 2 mins at 80° C. After cooling to 

 room temperature, the coloured material was extracted twice with 10 ml 75 % acetone for 20 mins. 

 The combined extracts were collected and the colour was estimated at 525 m/t. 



Estimation of ammonia 



After incubation, the Warburg vessels were dismantled, and 0.2 ml 1.5 M H2SO4 on filter paper 

 was inserted into the center well, and 0.3 ml saturated KgCOg added to the side arm. After thermal 

 equilibrium, the KjCOj was tipped and the vessels incubated with shaking at 37° C. After 3 hours the 

 filter papers were removed and the centre welis washed thoroughly with distilled HjO. The filter paper 

 and washings were collected in volumetric flasks, and aliquots were taken for NH4 determinations 

 (Braganca, Quastel and SchucherIsj. 



Pyruvate determination 



The method of Jowett and Quastel^' using 2 : 4 dinitrophenyl-hydrazine was adopted. 



Lactate determinations 



The method of Barker and SummersonI^ as modified by Umbreit^^ was used. 



Sulfanilamide determinations 



The method of Braxton and Marshall^o was used. Acetyl sulfanilamide was determined by 

 difference. 



Manometric experiments 



For measurements of COg evolution, 0.028 M NaHCOj was used as buffer at a final pH of 7.0. 

 The buffer was gassed with 7% COg + 93% Ng before use. The vessels were also gassed with this gas 

 prior to measurements of COg evolution. After gassing and thermal equilibrium (5 min) at 37° the 

 substrate was tipped in. All substrates were neutralised, when necessary, with caustic soda. For 

 oxygen absorption, in aerobic experiments 0.02 M sodium phosphate buffer at pH 7.3 was used. 0.2 

 ml of 20% KOH was placed in the centre well to absorb the COg. Air or Og formed the gas phase. 



Hydrogen absorption 



For hydrogen absorption, 0.02 M sodium phosphate buffer at pH 7.3, with KOH in the centre 

 well, was used. 



The vessels were gassed with nitrogen for five minutes and then with hydrogen for a further 

 five minutes after which thermal equilibrium was allowed to take place. 



Ferricyanide as terminal hydrogen acceptor 



The technique of Quastel and Wheatley^i was followed. 

 References p. 120. 



