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92 D. Lemos et al.rAquaculture 186 (2000 89–105
In the present study, original data on trypsin activity of larval Litopenaeus schmitti
and the proteinase pattern in SDS-PAGE of adult Farfantepenaeus paulensis, L.
schmitti, Farfantepenaeus californiensis, Litopenaeus Õannamei are provided. Larval L.
schmitti were fed with Chaetoceros calcitrans Ž80,000 cells ml y1 . and artificial
plankton ŽNippai Shrimp Feed, Japan; 0.03 mg larva y1 day y1 , 30 mm particle size .
from the stage protozoea I ŽPZ I . Freshly hatched Artemia sp. 15 nauplii larva y1
Ž
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day y1 , origin: Great Salt Lake . were added to the diet at protozoea III PZ III . After
.
Ž
metamorphosis to postlarva, the diet was composed of microalgae with increasing
amounts of artificial plankton and brine shrimp nauplii. The digestive glands of three to
five not sexually mature adult females of F. paulensis, L. schmitti, F. californiensis and
L. Õannamei were extracted, frozen at y208C and freeze dried. The animals were fasted
for 24 h before digestive gland extraction. F. paulensis and L. schmitti were previously
fed with frozen mussel and squid, while F. californiensis and L. Õannamei were fed a
prepared feed ŽPiasa Camaron La Paz, Mexico containing 35% protein, 8% lipid, and
´
.
4% fiber.
2.1. Protein and proteinase composition

Enzyme preparations, 0.02 mg of protein in up to 20 ml of sample buffer were loaded
per lane, in twin polyacrylamide gels, in a temperature controlled Ž48C electrophoresis
.
device. Gels of 8=10 cm are fast and have the ability to separate protein extracts.
Molecular weight standards Ž0.0175 mg were included on each plate. Electrophoresis
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was done at a constant current, 15 mA per gel. The separation should proceed for 1 to
1.5 h, when the tracking dye will reach a centimeter above the end of the plate. After
electrophoresis, one of the two identical SDS-PAGE gels is stained for protein with a
solution containing 40% ethanol, 10% acetic acid, and 0.1% Coomassie brilliant blue
R-250. The twin gel is immersed in 3% casein in 50 mM Tris–HCl, pH 7.5, for 30 min
at 58C, to allow the substrate to diffuse into the gel at low enzyme activity. Afterwards,
the temperature is raised to 258C for 90 min, for digestion of the protein substrate by the
active fractions. The gel is then washed in water and immediately fixed and stained by
immersion in the staining solution. After 2 h staining period, the gel is destained with
the same solution without the Coomassie dye, and dried. The gel stained for protein
gives the protein pattern of the samples Žproteinogram and the gel stained for activity
.
gives the pattern of enzyme activity Žzymogram . Clear zones on a blue background
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indicate proteinase activities and by comparison with the molecular weight standard
˜
bands, the molecular weight of the proteins or proteinases is obtained ŽGarcia-Carreno et
al., 1993 .. The molecular weight of active bands can be determined by regression
between the distance of molecular weight standard bands from the top border of the gel
Žcm and the log of their molecular mass kDa . The zymogram is more sensitive than
.
.
Ž
the proteinogram and usually bands of enzyme activity are observed where no bands of
protein are seen. A schematic sequence of these procedures is shown in Fig. 1a.
2.2. Proteinaceous proteinase inhibitors
To evaluate the presence of a proteinaceous proteinase inhibitor for a particular
proteinase in complex samples such as legume seed meals, an aqueous protein extract is