Graduate Thesis Or Dissertation
 

Partial purification of cathepsins from salmon muscle

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  • Cathepsins are intracellular proteinases that hydrolyze the peptide bonds of proteins. These enzyme have been implicated in the tenderization of aging beef, with the deterioration of radiation-stabilized meats on storage, and in the spoilage of fish prior to processing. Hence, the cathepsins of edible muscles are of concern to the food scientist. The purpose of the research reported herein was to develop procedures for the purification of the cathepsin from salmon muscle. The availability of a purified preparation of salmon muscle cathepsins should stimulate interest and research in the characterization of these enzymes and lead to better means for the control of catheptic activity in fish muscle. Results from these investigations indicate that salmon muscle cathepsins exhibit pH optima at 3.7, 6.9, and 8.5 when Folin's reagent was used to determine the products of protein hydrolysis; whereas, pH optima at 3.7 and 7.3 were obtained when the products of protein hydrolysis were determined by absorption at 280 mμ. Possible reasons for the differences in pH optima are discussed. It was decided to attempt the purification of the cathepsin optimally active at pH 3.7. The stability of this enzyme was found to be maximal at pH 6.5. The purification was accomplished by extracting the salmon muscle cathepsin with two parts 0.2 N KC1. The pH of this crude extract was adjusted to 5.5 and the precipitated proteins were removed by centrifugation before the pH of the supernatant was readjusted to the original pH of the extract. Upon dialysis of this fraction against 0.005 M phosphate (pH 6.5) a precipitate formed and was removed by centrifugation. The catheptic activity was precipitated from the surpernatant at 0.50 saturation with (NH₄)₂SO₄. The precipitate was recovered by centrifugation, dissolved in 0.005 M phosphate (pH 6.5), and dialyzed against the same buffer. A precipitate formed and was removed by centrifugation. This fraction was placed on a 2.5 x 35 cm column of DEAE-cellulose equilibrated with the starting buffer (0.005 M phosphate at pH 6.5). A concave concentration gradient was used to elute the proteins from the ion-exchange resin. Final buffer was 0.005 M phosphate (pH 6.5) containing 0.5 N NaCl. The absorbance (280 mμ) of the column effluent was continuously recorded and 10 ml fractions of the effluent were collected. Fractions comprising the various protein peaks were combined and concentrated by lyophilization. Two catheptic enzymes appeared to be separated by this procedure with maximum purification of 117 fold with 6.8 per cent recovery. During the development of the procedure, it was found that the fractions obtained from column chromatography could not be successfully concentrated by ultrafiltration, pervaporation, or "Aquacide #2" and that the presence of cysteine in the eluting buffer was not beneficial.
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