A large-scale procedure for the purification of a carboxylesterase present in the skin of oranges is described. The starting material was an acetone powder of the peel and the procedure involved ion-exchange chromatography, ammonium sulphate precipitations and gel filtration. Starting from 25 kg acetone powder, a yield of 26 mg of enzyme was obtained. This corresponded to a level of 1 mg of enzyme per case of 120 oranges. The preparation had a specific activity of 1 mkat/1./A280 and represented a purification of 6000-fold of the crude extract of the acetone powder.
The equivalent weight of the purified enzyme was determined by titration with both diethyl p-nitrophenylphosphate and [32p]diisopropylphosphorofluoridate . Reaction with either titrant was some three orders of magnitude slower than the corresponding reactions with the animal carboxylesterases and this allowed the first direct observation, using standard spectrophotometric techniques, of a biphasic "burst" of p-nitrophenol given by a carboxylesterase with diethyl p-nitrophenylphosphate. The equivalent weight range for the two methods was 65,800 - 69,100. This value contrasted with the apparent molecular weights of 41,300 and 43,000 obtained from zonal gel filtration and sedimentation equilibrium analysis respectively. Data obtained from electrophoretic and dilution experiments suggested that the preparation was a mixture of active and inactive forms.
The sequence of amino acids at the active site of the enzyme was investigated using [32P]diisopropylphosphorofluoridate to label the essential seryl residue. Comparisons of radioautographs of ionograms prepared from partial acid hydrolysates of [32P]diisopropylphosphoryl orange skin carboxylesterase and [32P]diisopropylphosphoryl chicken liver carboxylesterase confirmed that an amino acid near the active serine was substituted in the orange skin enzyme. Due to the scarcity of the orange skin enzyme, the phosphopeptides present in a partial acid hydrolysate of [32P]diisopropylphosphoryl chicken liver carboxylesterase were purified, identified and correlated where possible with the ionophoretic pattern of the orange skin enzyme. It was concluded that the amino acid sequence at the active site of the orange skin enzyme was X-Serine-Alanine where X was a nonpolar residue.
A peptic digest prepared from 10 mg of [32P]diisopropylphosphoryl orange skin carboxylesterase was purified and the peptides analysed. The results suggested that tryptophan was the substituted nonpolar residue.
Because of conflicting reports in the literature, the active site of bovine caudate acetylcholinesterase was compared with that of chicken liver carboxylesterase using the ionophoretic method described above. The sequence was shown to be Glutamic Acid-Serine-Alanine.