Electrophoretic characteristics of plasma alpha-1-proteinase inhibitors in healthy and mastitide-diseased sheep

The present study has been aimed at the identification of alpha-1-proteinase inhibitors in the blood plasma of the healthy and mastitide-diseased sheep of the Improved Valachian breed by means of electrophoresis analysis. In the experiment were inserted ten healthy and seven mastitide-diseased sheep, which come from submontane territory of the Presov district (eastern part of the Slovak Republic). From the milk of mastitide-diseased sheep have been isolated three species of bacteria and their combinations: Staphylococcus aureus beta-hemolyt. str. + Staphylococcus aureus, Streptococcus, uberis, St. aureus, S. uberis + Escherichia coli, St. aureus beta-hemolyt. str. + St. aureus. Two sheep of the diseased group suffered from the latent and five sheep from chronic mastitis. Blood was collected from the vena jugularis into he-parin (15 IU.ml/1). Prior to the analysis, blood plasma was incubated for 24 hours at 25 deg C with neuraminidase. Isoelectric focusation has been carried out on poly-acrylamide gels (230 x 160 x 0.5 mm) with ampholytes pH 4.2 - 4.9, 4.5 - 5.4, and 3.5 - 9.3 (T = 7.5 %, C = 3 %). The cathodic wick stripes contained 0.5M NaOH and anodic wick stripes contained 0.5M CH3COOH. The electrophoresis itself took place under the following conditions: 480 V/50 mA/25 W for 7 minutes, till the samples had soaked into gel. Electrophoresis continued for 80 minutes at 480 V/50 mA/25 W and the last 20 minutes at 1030 V/55 mA/25 W. The detection of alpha-1-proteinase inhibitors of trypsin has been done by modified method. The synthetic substrate of N-acetyl-DL-phenylalanin-(3-naphtylester) and enzyme trypsin (EC 3.4.21.4) were used. The reactions with trypsin were done using 0.1 M of phosphate buffer at pH 8.2 and at the temperature of 37.5 deg C for 20 minutes which has been dissolved in dimethyl sulphoxide. The results of alpha-1-proteinase inhibitors of trypsin after one-dimensional electrophoresis showed differences between healthy and mastitide-diseased sheep. In this study, alpha-1-proteinase inhibitors of trypsin with dominant trypsin activity in healthy sheep were on scale of isoelectric points pH 3.75 - 6.0. Protein fractions with lower inhibitory activity were in the zone of pH 3.5 -3.75 in mastitide-diseased sheep. There was registered the lowest activity and number of protein fractions of trypsin inhibitors in diseased sheep. Described results may be a good contribution to prevention of the disease of sheep in connection with the environment and breeding intentions.