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6209028 
Book/Book Chapter 
[46] Hippuric acid synthesis: Benzoic acid+Glycine→CoAATPHippuric acid 
Chantrenne, H 
1955 
1 
Methods in Enzymology
ISSN: 0076-6879
EISSN: 1557-7988 
Academic Press 
Methods in Enzymology 
2 
346-350 
Publisher Summary This chapter discusses about the hippuric acid synthesis. T h e enzymes involved are associated with large cytoplasmic particles. They have been obtained in solution and partially purified. Hippuric acid synthesis from glycine and benzoate involves at least two steps: (a) benzoyl activation with the formation of S-benzoyl-CoA at the expense of some source of energy; (b) condensation of benzoyl-CoA with glycine. In the course of the assay method, hippuric acid is extracted from its acid aqueous solution in an organic solvent which does not extract free glycine. The extracted hippuric acid is hydrolyzed and glycine determined in the hydrolyzate. Ether is extracted and glycine is determined by formol titration. The assay method as described can be applied directly to mixtures containing soluble enzymes. The addition of sulfuric acid blocks the enzyme reaction, and after shaking with the extraction fluid and centrifuging, the flocculated proteins form a thin, compact layer between the two liquid phases, which does not interfere with the assay. For crude homogenates, it is necessary to remove the proteins first. Thus to the incubation mixture add 0.2 vol. of 10% metaphosphoric acid solution, then centrifuge and determine hippuric acid in the supernatant. It is recommended that the recovery of hippuric acid be checked in each case by adding known amounts of hippuric acid to the tissue preparation and determining hippuric acid immediately.