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94 D. Lemos et al.rAquaculture 186 (2000 89–105
Table 1
Substrates and inhibitors commonly used for shrimp proteolytic enzyme assays
Enzyme Substrate Inhibitor
Total proteinases Azocasein or casein a
Trypsin TAME Ž Na-p-toluenesulphonyl-L- TLCK Žtosyl-lysine chloromethyl
Arg methyl ester . b ketone . c
BAPNA Ž Na-benzoyl-DL-Arg-p-
nitroanilide . d
Chymotrypsin SAPFNA Ž N-succinyl-L-Ala-L-Ala-L-Pro- ZPCK Žcarbobenzoxy-
L-Phe-p-nitroanilide . e phenylalanine chloromethyl ketone . c
Serine proteinases PMSF Žphenylmethylsulphonyl
fluoride . c
a
˜
Garcia-Carreno et al. Ž1993 .
.
b
Rick Ž1984 . .
c
Beynon and Salvesen Ž1989 . .
d
.
Erlanger et al. Ž1961 .
e
DelMar et al. Ž1979 .
.
inhibition cannot be measured by visual observation of PAGE zymogram and results are
interpreted as presence or absence of casein digestion. Casein is used as substrate
because it is readily digested by penaeid proteinases such as trypsin and chymotrypsin
ŽJiang et al., 1991; Le Moullac et al., 1996 . This technique detects only proteinases that
.
form short chain polypeptides. Peptidases Žor exopeptidases are not expected to
.
generate clear zones in the blue background.
2.4. Quantification of proteinase actiÕity
Total proteinases, trypsin and chymotrypsin activities are usually determined by the
incubation of enzyme extract with natural or synthetic substrates ŽTable 1 at controlled
.
temperature and pH. As the substrate is hydrolyzed, changes in absorbancy can be
detected at specific wavelengths. Azocasein is a commonly used substrate for the
˜
quantification of total proteinase activity ŽGarcia-Carreno et al., 1993 . Ten microliters
.
of enzyme extract are added to 0.5 ml of 1.5% azocasein in 50 mM Tris–HCl buffer, pH
7.5 ŽGarcia-Carreno, 1992a at 258C. The reaction is stopped 10 min later by the
˜
.
addition of 0.5 ml of 20% trichloroacetic acid ŽTCA , and the mixture is centrifuged for
.
5 min at 6500=g. The supernatants are separated from the undigested substrate and the
absorbance at 366 nm for the released dye recorded. The assay must include appropriate
blanks as internal positive and negative controls. Trypsin and chymotrypsin activities are
determined by the rate of hydrolysis of synthetic specific substrates. Trypsin activity can
be measured by using Na-p-toluenesulphonyl-L-Arg methyl ester ŽTAME or Na-
.
benzoyl-DL-Arg-p-nitroanilide ŽBAPNA substrates. Samples of 300 ml of enzyme
.
extract are mixed with 1.2 ml of 10 mM TAME in 46 mM Tris buffer, pH 8.1,
containing 11.5 mM CaCl at 258C, and the change in absorbancy at 247 nm is recorded
2
for 3 min ŽRick, 1984 . One millimolar BAPNA is dissolved in 1 ml dimethylsulfoxide
.
ŽDMSO and then made to 100 ml with Tris–HCl, pH 7.5, containing 20 mM CaCl .
.
2