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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

1.3310-7

M (6.1810-3

mg/ml).

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Table 1. Analysis of yield and specific activity

Fraction:ProteinConcentratio

n (mg/ml):

Total

Protei

n(mg):

Percent ofTotal

Protein:

Activity ofEach Fraction

(mol/sec/ml):

Total

Activity

(mol/sec):

Specific

Activity

(mol/sec/mg):

F1 17 935 100 5.09 280.01 0.299F2 2 72 7.700 3.23 116.33 1.615

ASI 7.7 24.64 2.635 1.83 5.86 0.238

ASII 2.6 5.98 0.610 22.51 51.77 8.658

ASIII

(desalted)0.05 3.95 0.422 0.033 9.10 0.664

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.

y = -0.042x + 0.6409

R = 0.9994

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60 70 80 90

Absorbance

Time (s)

ASII (100X)

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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.

y = 1.0148x - 0.1329

R = 0.9945

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Concentration(mg/ml)

Absorbance

BSA Standards

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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).

y = -0.0069x + 0.6244

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60 70 80

Absorbance

Time (s)

Low Salt Peak

y = -0.0003x + 0.6313

0.605

0.61

0.615

0.62

0.625

0.63

0 20 40 60 80 100

Absorbance

Time (s)

High Salt Peak

A.

B.

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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 8

L of the molecular weight standard or 20

L of a 2mg/ml dilution of thecorresponding 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.

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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.

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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).