5. ANÁLISIS INTERNO
5.6. MATRIZ DE EVALUACIÓN INTEGRADA DE LA SITUACIÓN INTERNA
LANE:
1 -5 : A280 peaks from elution of phenyl superose column stained with coomassie blue
R250. Lane 3 contains the active fraction shown in lane 8 of Figure 7.7
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LANE: 1
2
3
4
5
6
7
8
97.9 : SDS-PAGE of Pu rified Protein
Protein purified from hydrophobic interaction chromatography was run on an SDS gel and stained with coomassie blue R250
LANE:
1 : Biorad molecular weight standards
2-4 : Pure Cytosolic AlDH
5-8: Purified Novel Protein
9 : Biorad molecular weight standards
97.4 kDa
3 1 .0 kDa +-- 2 1 .5 kDa
7 - 1 69
LANE :
I
2
3
4
7. 10:
Western Blot of Pu re Novel P rotein and AIDH I
Western Blot performed as outlined in section 2.2. 1 5, using 4-chloro- l naphthol as substrate
LANE:
1 , 3 : Pure Novel Protein (2 different Loadings) 2,4: Pure AlDH 1 (2 different loadings)
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7.2.7 HPLC
Size
Exclusion ChromatographyAfter preparative isoelectric focusing, there were only 5-10 proteins left in the mixture (depending on the success of this step) from which to isolate the desired protein. At this stage, size exclusion chromatography or gel filtration is a good option for further purification. This technique separates a mixture of proteins primarily on the basis of their Stokes radius, which in turn depends on the native molecular weight and on the overall shape of t�e protein. The columns used were Superose-1 2 and Superdex-75 (Pharmacia) under the control of a SMART HPLC (Pharmacia) system consisting of SMART manager software, J..1Precision pump, variable volume gradient mixer, J..1Separation unit, J..1Fraction collector and J..1Peak monitor, connected to cooling apparatus. The Superdex 75 column (PC 3 .2/30), has a 2.4 m1 volume, 3.2 x 3 00 mm dimensions, 1 3 IlM particle size and a molecular weight range from 3-70 kDa. The Superose 1 2 (PC 3 .2/30) size exclusion column has a 2.4 ml volume, 3 .2 x 300 mm dimensions, 1 0 IlM particle size, and a molecular weight range from 1-300 kDa. The buffer used was 200 mM Hepes pH 7.2, 1 0 % (w/v) glycerol, 0.3 mM EDTA, 0.5 mM DTT, and 1 0 mM NaCl. Each run was at 40 Ill/min,. 1 0 °C, collecting 80 Illfractions, and proteins were identified by measuring the absorbance at 280 nm (A2so).
Size exclusion chromatography usmg a Superose-1 2 column (separation range 1-300 kDa) gave adequate separation of two major protein peaks (Figure 7. 1 2), although the mixture of 5-10 proteins contained in the initial sample did not resolve very well (Figure 7. 1 3, lane 9), indicating that the native (or eff1ective) molecular weights of these proteins were too similar for this column to separa.te. The protein of interest also retained activity, which was consistently found in the first peak to elute from the column. SDS-P AGE of the sample after preparative isoelectric focusing and the peaks separated by gel filtration showed that the peak containing the activity, though symmetrical, contained a minimum of 2 bands, one at 40-45 kDa and one at 25-28 kDa (Figure 7. 1 3). The band at 25-28 kDa often resolved as a doublet. The protein of interest was thought to be the 40-45 kDa band, due to the fact that the first successful purification had identified a band ofthis size (Figure 7.9). Further purification attempts concentrated on isolating this band ..
To try to separate the proteins more effectively, a Superdex-75 column was used. This column has a separation range of 3-70 kDa, and a slightly larger particle size of 1 3 J..1M, although the diameter and length dimensions were identical to the Superose- 12. This column may separate a range of proteins within this molecular weight range better than
D iscard pellet Discard pellet Discard pellet Discard supematant 7-1 7 1
Homogenise 1 kg fresh sheep liver Spin 9000 x g 15 min Supematant Spin 13000 x g 1 5 min Supematant PEG 8000 to 1 0% (w/v) Spin 9000 x g 3 0 min Supematant PEG 8000 to 20% (w/v) Spin 9000 x g 30 min Pellet Redissolve in Bis-tris pH 6.2
t
D EAE-Iontosorb Initial wash-throughPreparative isolectric focusing Active fraction
FPLC phenyl Superose Active fraction
Pure Prottein
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the Superose-1 2 . However, this did not turn out to be the case. Elution profiles using both columns looked very similar (Figure 7 . 1 2, 7. 1 4) . Although activity was maintained throughout the process of gel filtration using both columns, separation of the protein of interest from the other proteins could not be achieved (Figure 7. 1 3 , 7. 1 S) . To estimate the molecular weight of the protein, the elution volume of the protein was compared to those of standards of known molecular weight using the Superdex-75 column (Figure 7. 1 6). The buffer system used was 5 0 mM Hepes, pH 7.4, 20 mM NaCI, O . S mM EDT A, 1 0 % (v/v) glycerol. Standard proteins used were sheep liver AlDH 1 (MW
220 kDa), bovine lactoferrin (80 kDa), bovine serum albumin (BS A) (66 kDa), �-lactoglobulin (3 S kDa), and a-lactalbumin ( I O- l S kDa). The estimate obtained from size exclusion chromatography (Figure 7. 1 7) of the molecular weight of the unknown protein was 40-S0 kDa, which is a very similar size to the band seen on SDS-P AGE. From Figure 7 . 1 6 and SDS-P AGE data, it was concluded that the protein had a native molecular weight of 42-45 kDa, and a monomer size of 42-4S kDa, and therefore was a monomeric protein. This raised interesting questions. AlDH proteins are typically dimers or tetramers of 50-SS kDa subunits, while ADH proteins are typically dimers or tetramers of �40 kDa subunits (Bosron et al. , 1 993). The elucidated characteristics of the protein did not seem to fit into either of these categories, which made the isolation and identification of the protein vital, as it appeared to be a novel protein with an alcohol dehydrogenase activity. Monomeric ADH proteins have been identified in microbes, e.g. ethanol dehydrogenases from Acetobacter aceti and A. polyoxogenes, though all identified mammalian ADHs are dimeric (Reid and Fewson, 1 994).
7.2.8
Hydroxyapatite ChromatographyHydroxyapatite resin consists of an inert matrix to which calcium groups are attached. The bases of the attraction of proteins to hydroxyapatite resins are both non-specific attraction between protein positive charges and hydroxyapatite, and specific complexing of protein carboxyl groups with the calcium loci (Gorbunoff & Timasheff, 1 984). Elution of bound proteins can be either by non-specific ion charge elution, or by specific displacement of protein groups from sites on the column to which they have complexed. The initial buffer used was 10 mM NaH2P04, pH 7.0, containing 0.3 mM DTT and 0.5 mM EDT A, and to elute the proteins a series of increases in the phosphate ion concentration - 75 mM, 1 00 mM, I SO mM, 200 mM and 400 mM was used. Initially a batch trial was performed. Partially purified protein was added to 0. 5 g ( 1 . 5 ml when
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I 7. 12: HPLC Size Exclusion of Purified ProteinThe elution profile is created by continuously monitoring the A280. The absorbance scale is approximately 0-0 . 5 absorbance units.
Putative Protein
LANE: 1 2 3 4
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5 6 7 8 9
7. 1 3 : SDS-PAGE of HPLC Size Exclusion Fractions 974 kDa 66.2 kDa 45.0 kDa 3 1 .0 kDa 2 1 . 5 kDa 144 kDa
These fractions relate to the Superose- 1 2 size exclusion chromatography el ution profile shown in Figure 7. 1 2
LANE:
1: Fraction 9 (No activity) 2: Fraction 10 (Active) 3 : Fraction 1 1 (Acti ve)
4: Fraction 1 2 (No activity)
5: Fraction 1 3 (No activity)
6: Fraction 14 (No activity) 7: Fraction 1 5 (No activity)
8: Biorad molecular weight standards
o..su 1.45 1.40 1.35 lUO US 120 a.15 1.10 a..oS 1.00 AU 0.0 7- 1 75
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[ ... 5 2.5 Id Active fractions 7. 1 4 : HPLC Size Exclusion of Purified ProteinThe elution profile is created by continuously monitoring the A280. The absorbance scale is approximately 0-0 . 5 absorbance units.