Partial characterization of canine neutrophil elastase

SDS, 21 mM EDTA, pH 7.3). The samples were prepared by adding 24 µl of sample to 4 µl of reducing agent and 10 µl of NuPAGE^® SDS sample buffer (4 g sucrose + 0.68 g Tris-base + 0.67 g Tris-HCl + 0.8 g SDS + 0.006 g EDTA + 0.75 ml Serva blue G250 + 0.25 ml Phenol red + ultra pure water to 10 ml). Samples were incubated at 70°C for 10 min. After incubation 23 µl of each of the samples were loaded into the wells. In an addition to the samples a standard protein solution was loaded into the first lane. The standard protein solution contained 12 different proteins with a known molecular mass, including myosin: 200 kDa, β-galactosidase: 116.3 kDa, phosphorylase: 97.4 kDa, bovine serum albumin: 66.3 kDa, glutamic dehydrogenase:

55.4 kDa, lactate dehydrogenase: 36.5 kDa, carbonic anhydrase: 31 kDa, trypsin inhibitor: 21.5 kDa, lysozyme: 14.4 kDa, aprotinin: 6 kDa, insulin B chain: 3.5 kDa, and insulin A chain: 2.5 kDa. The electrophoresis gel was run at a constant voltage of 200 V for 35 minutes. Immediately after completion the gel was removed from the cassette and stained with a silver stain kit, according to the manufacture’s instructions.

3.2.2. Partial characterization of canine neutrophil elastase

sieving polymer matrix, the molecules are separated by size (the smaller the protein the faster it migrates). Proteins are then intercalated with dye molecules and are detected by laser-induced fluorescence. Data is translated into gel-like images (bands) and electropherograms (peaks). A standard curve of migration time versus fragments size is plotted with the help of a ladder (protein standards). From the migration times measured for each fragment in the sample, the size is calculated. Two internal markers are run with each of the samples bracketing the overall sizing range. The

“lower” and “upper” markers are internal standards used to align the ladder data with data from the sample wells. This is necessary to compensate for drift effects that may occur during the course of a chip run.

Quantitation is done with the help of the upper marker. The area under the upper marker peak is compared with the sample peak areas. Because the concentration of the upper marker is known, the concentration for each sample can be calculated.

The chip and the sample (purified cNE) were prepared according to the manufacturer’s instructions.

3.2.2.2. Estimation of isoelectric point

The isoelectric point was estimated by polyacrylamide gel electrophoresis using a NOVEX^® Pre-Cast vertical IEF gel, 5% polyacrylamide, and 2% ampholytes with a pH range of 3 to 10. Special protein markers for isoelectric focusing gels were used to generate a standard curve. The protein marker contained 9 different proteins, ranging from a pI 10.7 (cytochrome C, horse heart) to a pI 3.5 (amyloglucosidase, Aspergillus niger).

The sample was prepared by thoroughly mixing one part of sample with one part of NOVEX^® IEF sample buffer. The buffer for the cathode was prepared by adding one part NOVEX^® IEF cathode buffer to nine parts of deionized water. The buffer was degassed for 20 minutes and then filled into the upper buffer chamber. The anode buffer was prepared by adding 1 part of NOVEX^® IEF anode buffer to 49 parts of deionized water and poured into the lower buffer chamber. Samples were loaded into the wells and the gel was run at a constant voltage of 100 V for 1 hour, followed by a constant voltage of 200 V for 1 hour and finally at a constant voltage of 500 V for 30

fixing solution (17.3 g sulphosalicylic acid, 57.3 g trichloroacetic acid, in 500 ml deionized water). The gel was stained with a silver stain kit, following the manufacture’s instructions. A standard curve was generated by measuring the migration distance from the well to the protein bands of the markers.

3.2.2.3. Determination of the extinction coefficient for neutrophil elastase The extinction coefficient of cNE was determined based on the bicinchoninic acid (BCA) assay (Smith et al., 1985). The BCA™ protein assay is used for the colorimetric detection and quantification of total protein in a solution. The method is based on the reduction of Cu⁺² to Cu⁺¹ in an alkaline environment. The reduced copper forms a purple colored complex with bicinchoninic acid, which is colorimetrically measured at 542 nm. The absorbance of the complex is nearly proportional with increasing protein concentration.

The assay was performed according to the manufacturer’s instructions in a microplate format. Briefly, 8 standards from bovine serum albumin were prepared. The protein concentration of the standards ranged from 2,000 µg/ml to 25 µg/ml. Purified cNE with a known absorbance at 280 nm was diluted 1 in 2, 1 in 4, and 1 in 8 in PBS. The BCA™ working reagent was prepared by mixing 50 parts of BCA™ reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M sodium hydroxide) with 1 part of BCA™ reagent B (4% cupric sulfate). A volume of 15 µl of each standard, samples, and a blank was loaded into the microplate wells, and 200 µl of the previously mixed BCA™ working reagent were added to each well. The plate was incubated for 30 min at 37ºC, and the absorbance was measured at 562 nm.

The absorbance of each well was corrected for non-specific binding (blank). A standard curve was prepared by plotting the average of the duplicates for each BSA standard versus its concentration in µg/ml. The protein concentration of each unknown sample was interpolated from the standard curve

The extinction coefficient was calculated by dividing the previously measured absorbance of the pure protein solution at 280 nm by the protein concentration, calculated with the help of the BCA™ protein assay. Each of the 3 cNE dilutions (1 in 2, 1 in 4, and 1 in 8) were calculated and the mean was taken.

3.2.2.4. Material preparation for N-terminal amino acid sequencing

Twenty µg of purified cNE were transferred onto a PVDF membrane using the ProSorb™ sample preparation cartridge. The protein transfer was done according to the manufacture's protocol. The sample was analyzed at the Department of Biotechnology at the Jagiellonian University and Bio Centrum Ltd. facility in Kraków, Poland. The N-terminal amino acid sequence analysis of the first 25 residues of canine neutrophil elastase was obtained by use of the Edman degradation method.

During the Edman degradation method phenylisothiocyanate reacts with the amino acid residue at the amino terminus to form a phenylthiocarbamoyl derivative.

Trifluoroacetic acid is used to cleave off the first amino acid as its anilinothialinone derivative, followed by the conversion to a phenylthiohydantoin derivative. Bound to phenylthiohydantoin the amino acid is then detected by high-performance liquid chromatography (HPLC). This process was repeated sequentially until the first 25 amino acid residues were determined.