5. ANÁLISIS INTERNO
5.5. ANÁLISIS COMERCIAL Y DE VENTAS
252.59
g(5)
220.2
Evaporation to the correct final weight took approximately
8
hours. When the correct weight was reached, the gel was connected to the LKB power supply, and proteins separated at8
W constant power at10ce
for14-16
hours. Separation was easily visualised by looking at the banding pattern produced after focusing, compared to a uniform red colour before focusing (Figure 7. SA). After separation, the fractionation grid was placed into the gel (Figure 7. SB). The grid separates the gel into 3 0 even sections, which can be removed separately from the tray. The pH of each fraction was then measured, and proteins separated from the gel by resuspending the gel in a small amount of50
mM tetrasodium pyrophosphate buffer, pH9,
and passing the fraction over glass wool. The activity of each fraction was then measured by analytical IEF with activity staining, as described previously.A good purification was achieved using this technique. Of primary importance, active protein was present after evaporation and focusing which took up to
24
hours (Figure 7.6). The only fraction in which activity was seen was typically the fraction nearest the cathode, which had a measured pH of-8.8.
The result obtained using preparative isoelectric focusing was-2
rnl of active protein, containing an enrichment of the protein of interest. At this stage, no further analysis was performed on this mixture, as it was deemed to be more important to retain as much active protein as possible for the next step of purification.7-1 62
7.2.6
FPLC Hydrophobic Interaction ChromatographyTo further purifY this protein, hydrophobic interaction chromatography was the next step used. Separation using this technique is based on the differing surface hydrophobicity of proteins, such that proteins will interact with a phenyl group (or other hydrophobic group) attached to a matrix according to the amount of hydrophobic patches on their surface. Proteins bind to the column under conditions of high salt, and are eluted by decreasing the salt concentration of the buffer. Hydrophobic interaction chromatography is a non-specific technique, and does not usually give sharp separations (Scopes, 1 987c). However the capacity is high and often good recoveries are achieved. The column used
in this case was a phenyl-Superose column, dimensions 5 x 5 0 mm (pharmacia),
connected to a Pharmacia FPLC system (LCC-500), a Pharmacia single path
UV-l
monitor, and a Kipp and Zonen BD-4 1 chart recorder. Proteins were identified by
measuring the absorbance at 280 nm . The column was equilibrated by passing through
20 mM phosphate buffer pH 7.2, containing 1 M ammonium sulphate, for 25 minutes at 0 . 5 m1!min. After the column had been equilibrated, the protein solution was filtered through an 0.22 !J.M filter prior to injection. A linear gradient was then run from 1 M ammonium sulphate to 0 M ammonium sulphate over 60 min at 0 . 5 m1!min, and then held at 0 M for a further 3 0 min. Fractions were collected as proteins were eluted from
the column by A280 peaks on the chart recorder. Fractions were assayed for activity as
previously described using the IEF activity assay, and in addition the protein content was
assessed by staining an analytical IEF gel with Coomassie blue R250. The results show
that the protein of interest was active, and protein staining of the IEF gel showed that the protein was pure (Figure 7.7, 7. 8). To further check the purity of the protein, SDS PAGE was performed on the sample as described previously (section 2 . 2 . 8) (Figure 7. 9). Although a small impurity could be seen, the protein was estimated to be about 90 % pure. The monomeric molecular weight was estimated to be 40-45 kDa by comparison to SDS-7 protein size standards (Sigma).
Western blot analysis was performed on this sample using antibodies raised to sheep liver AlDH 1 as described in section 2.2. 1 5 (Figure 7. 1 0). The antibody did not interact with the purified protein, which helped to confirm our revised hypothesis that the isolated protein was not a member of the aldehyde dehydrogenase family, and supported the idea
that it was in fact an alcohol dehydrogenase. A portion of the remainder of the purified
protein was subjected to automated N-terminal sequence analysis as described in section 2.2. 12. No sequence data were obtained. This is likely to have been due to one of two
A
B
7- 1 63
A: Gel slurry after focusing for 1 4- 1 6 hours as described in the text, showing the characteristi c banding pattern
7- 1 64
Gel Artefact
New Activity
LANE: 1 2 3 4 5 6 7 8
7.6 :
IEF Gel of IsoelectricFractions
Preparative IEP was run as described in section 7.2. 5 . The focused gel was then fractionated using the metal grid, the protein extracted from each section, and run on the analytical IEP gel above. The gel was stained for activity as described.
LANE:
1 : Active before pIEP
2: pH 7.5 fraction 3 : pH 7.6 fraction 4: pH 7.8 fraction 5 : pH 8.0 fraction 6: pH 8.2 fraction 7: pH 8.4 fraction 8: pH 8.8 fraction
7-1 65
was N-terminally blocked. The remammg protein was cleaved usmg the chemical cyanogen bromide as described in section 2.2. 1 1 . 1 . Cyanogen bromide cleavage was chosen because it is likely to produce fewer fragments by cleaving at methionine residues than, say, by cleaving at arginine and lysine residues by trypsin. Peptides generated were separated by HPLC as described in section 2.2. 1 1 . 6. Two large peaks were trapped, dried down completely under vacuum, and sent for automated sequencing as before. No sequence was obtained.
At this stage the main objective of this section of the project had been achieved. The putative novel protein had been purified. However, the identification of the protein was unclear. From the available data, isoelectric point (�8. 5), subunit molecular weight (40-45 kDa), tissue and organism origin (sheep liver), western analysis (negative AlDH interaction), and activity (alcohol dehydrogenase), it seemed likely that the protein was a previously unidentified alcohol dehydrogenase isoenzyme. To confirm this hypothesis, the most important information needed was some unambiguous sequence which could be compared with other identified alcohol dehydrogenases. In addition the native molecular weight may provide some information as to the identity of the protein. In order to achieve these aims, more pure protein would need to be generated.
Multiple repetitions of the protocol outlined in Figure 7. 1 1 did not yield any pure protein. This was puzzling, considering that the method had previously yielded pure
protein. The step which was not behaving as expected was the final stage of
hydrophobic interaction chromatography, despite the fact that the system and all components had not been altered from that which had previously been used. A major problem in the unsuccessful runs was that the protein did not seem to be binding to the column. In the first successful run, the activity was retained on the column, and was found in the final elution peak, indicating that the protein was quite hydrophobic. However, in subsequent runs the activity was found in the first peak to elute indicating that the protein was interacting only weakly or not at all with the column. Different conditions were tested to see if altering the pH or the ionic strength of the buffer system would allow binding of the targeted protein to the column in an effort to separate it from other contaminating proteins. None of these efforts were successful. It was necessary therefore to try alternative methods of obtaining pure protein after the preparative isoelectric focusing step.
7- 1 66
LANE :
1
2
3 4 5 6 7 8 9Gel Artefact
New Activity
7.7: IEF Gel of FPLC Interaction
Fradions
IEF of fractions separated by phenyl superose hydrophobic interaction chromatography. Each protein peak was identified by the A280, and tested for the activity of interest. Activity was found in the fraction in lane 8 .
LANE
1: Active sample before plEF
2: Pooled plEF fraction 3 -9: A280 peaks from
elution of phenyl superose column. 8: Acti ve A280 peak.
7- 1 67
LANE:
1
2
3
4
5
7.8: