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David McCarthy BIOL 272-53 Purification of glutamate-aspartate aminotransferase from porcine heart Abstract: Glutamate-aspartate aminotransferase (GOT) is an enzyme

that catalyses production of glutamate and oxaloacetate from -ketoglutarate and aspartate. GOT plays a key role in nitrogen metabolism. In this experiment, GOT was analyzed in pig heart. Homogenized pig heart extract (F1) was heat treated with -ketoglutarate and maleate to precipitate out heat unstable proteins. The remaining heat-stable extract (F2) was fractionated with ammonium sulfate into three fractions (ASI, ASII, and ASIII). ASIII was dialyzed to remove ammonium sulfate. GOT activity in each fraction was determined from a coupled assay with the malate dehydrogenase catalyzed conversion of oxaloacetate and NADH to malate and NAD+. The concentration of all proteins in each fraction was found using both tube and microplate BCA protein assays. Fraction ASII was purified with carboxymethyl cellulose cation exchange chromatography following equilibration with molecular exclusion chromatography. SDS-PAGE was used to find the protein composition of the fractions. ASII had a significantly higher specific activity than ASI and ASIII at 8.658mol/sec/mg, suggesting that a majority of the GOT enzyme was fractioned into ASII. The yield of ASII was 18.49 percent. SDS-PAGE displayed bands corresponding with the molecular weight of GOT in F1, F2, ASII, and faintly in ASIII, while ASI had too many bands merging in this region to distinguish a single band that could represent GOT. Assays of fractions from purified ASII had activities of .370mol/sec/ml and .00161mol/sec/ml for a low and a high salt wash respectively. The concentration of protein in the peak fraction of the high-salt wash was 1.3310-7M (6.1810-3mg/ml).

Table 1. Analysis of yield and specific activity Total Protein Percent of Activity of Protei Fraction: Concentratio Total Each Fraction n n (mg/ml): Protein: (mol/sec/ml): (mg): F1 17 935 100 5.09 F2 2 72 7.700 3.23 ASI 7.7 24.64 2.635 1.83 ASII 2.6 5.98 0.610 22.51 ASIII 0.05 3.95 0.422 0.033 (desalted)

Total Activity (mol/sec) : 280.01 116.33 5.86 51.77 9.10

Specific Activity (mol/sec/mg) : 0.299 1.615 0.238 8.658 0.664

ASII (100X)
0.7 0.6 Absorbance 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 70 80 90 Time (s) y = -0.042x + 0.6409 R = 0.9994

Figure 1. Coupled assay of ASII to indirectly measure GOT activity through absorbance of NADH at 340nm. ASII was diluted 100 fold. Absorption decreases as the GOT catalyzed reaction produces oxaloacetate.

BSA Standards
1.4 1.2 Concentration (mg/ml) 1 0.8 0.6 0.4 0.2 0 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Absorbance y = 1.0148x - 0.1329 R = 0.9945

Figure 2. Concentration versus absorbance of BSA standards for the BCA protein tube assay to determine the concentration of all proteins in each fraction. Presence of cysteine, cystine, tryptophan, and tyrosine residues under basic conditions causes the reduction of Cu2+ to Cu+. BCA reacts with Cu+ to form a colored chelate product that can be detected.

A.
0.7 0.6 Absorbance 0.5 0.4 0.3 0.2 0.1 0 0 10 20

Low Salt Peak

y = -0.0069x + 0.6244

30

40 Time (s)

50

60

70

80

B.
0.63 0.625 Absorbance 0.62 0.615 0.61 0.605 0 20

High Salt Peak

y = -0.0003x + 0.6313

40 Time (s)

60

80

100

Figure 3. Assays of low (A) and high salt (B) peak fractions from the carboxymethyl cellulose chromatography of ASII. ASII was equilibrated with .03M acetate through molecular exclusion chromatography before it could be purified. A corresponds with fraction 5 of the .03M acetate low salt wash (activity: .370mol/sec/ml) and B corresponds with fraction 15 of the .08M acetate high salt wash (activity: .00161mol/sec/ml).

Figure 4. SDS-PAGE gel of standards (A and G), F1 (B), F2 (C), ASI (D), ASII (E), and desalted ASIII (F). Each standard protein is labeled with the corresponding molecular weight. Wells were loaded with 8L of the molecular weight standard or 20L of a 2mg/ml dilution of the corresponding fraction (desalted ASIII was undiluted).

As expected, the percentage of total protein decreased with each successive fraction (Table 1). One of the limitations of the BCA assay was that total protein concentrations were determined by combining data from two microplate assays and two tube assays, each with fraction dilutions of approximately .5mg/ml and 2.0mg/ml. Final concentration values were calculated by averaging values from the four sets of data that appeared close together, while ignoring outliers (pg. 12 and 13 of notebook). This data could be improved by increasing the number of assays and testing a larger variety of dilutions.

During the purification of ASII with ion exchange chromatography on carboxymethyl cellulose, equilibrated ASII was run through a CMC column with a low salt buffer followed by a high salt buffer. Based on the absorbance at 280nm for each fraction, a majority of the GOT was eluted by the low salt wash (pg. 16 of notebook). Assays of fractions with the highest absorbance from each wash had activities of .370mol/sec/ml for the low salt wash and .00161mol/sec/ml for the high salt wash (pg. 17 of notebook). The specific activity increased from fraction F1 to fraction F2 (Table 1), suggesting that GOT showed greater heat stability in the presence of -ketoglutarate and maleate than other proteins in the pig heart homogenate. Among ASI, ASII, and desalted ASIII, ASII had a significantly higher specific activity than the other two fractions. This implies that when the heat-stable pig heart extract was fractioned based on sensitivity to ammonium sulfate, a majority of the GOT enzyme precipitated into ASII. The low specific activities observed for ASI and ASIII relative to ASII suggests that this was an effective separation scheme. However, the yield for ASII was low at 18.49 percent (pg. 21 of notebook), which indicates that this procedure could be improved. The molecular weight of porcine cytoplasmic GOT is 46,475 Da. There was a clear band in the SDS-PAGE gel for the F1, F2, and ASII fractions corresponding with this molecular weight (Figure 4 and pg 22 of notebook). The desalted ASIII fraction had a faint band at this molecular weight and ASI had too many bands merging in this region to distinguish a single band that could represent GOT. Desalted ASIII was loaded undiluted because the concentration was below the 2mg/ml requested in the protocol. The gel stuck to a glass plate of the gel mold and ripped into many pieces when it was removed. A majority of the fragments were pieced together and the breaks did not appear to significantly impact the results.

The extinction coefficient of porcine cytoplasmic GOT is 67630. Pyridoxal phosphate is a cofactor necessary for this enzymes activity. Based on the Beer-Lambert Law, the extinction coefficient, and the absorbance of .009 (pg. 16 of notebook) the concentration of protein in the peak fraction of the high salt wash of purified ASII was 1.3310-7M (6.1810-3mg/ml).