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. / Graduation date: 1965
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27005 |
| Date | 15 March 1965 |
| Creators | Ting, Chao-Yun |
| Contributors | Montgomery, Morris W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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