Separation and Purification of α Chymotrypsinogen and ... CHROM3… · When the packing process...
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Application Note: CHROM303 Introduction Chromatography Analysis For the comparison of OmniSep A-Octyl Dash and Octyl Sepharose FF the sample purified was a mixture of Cytochrome C, Lysozyme and α-Chymotrypsinogen. Cytochrome C proteins are found loosely associated with the inner membrane of the mitochondrion. Cytochrome C is a small (molecular weight about 12,000 Dalton’s), highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an essential component of the electron transport chain, where it carries one electron. It is capable of undergoing oxidation and reduction, but does not bind oxygen. Cytochrome C is a highly conserved protein across many species, found in animals, plants, and many unicellular organisms. Its structure consists of a chain of 100 amino acids. The Cytochrome C molecule has been studied in evolutionary biology; its amino acid sequence is conserved in mammals differing by only a few residues. For example, the sequences of cytochrome c in humans are identical to that of chimpanzees (our closest relatives). Lysozyme, also known as muramidase is a glycoside hydrolase enzyme that damages bacterial cell walls. Lysozyme is present in a number of secretions, such as tears, saliva, human milk, and mucus and large amounts of lysozyme can be found in hen egg white. Chymotrypsinogen is a proteolytic enzyme and is a precursor of chymotrypsin a digestive enzyme. It is constructed of a single polypeptide chain consisting of 245 amino acid residues and is synthesized in the cells of the pancreas and stored. The cell when stimulated by either a hormonal signal or a nerve impulse empties the stored contents into a duct leading into the duodenum. Chymotrypsinogen must be inactive until it gets to the digestive tract. This prevents damage to the pancreas and other organs. Two identical columns were used for the evaluation, separation and purification. They were packed in the OmniSep manufacturing suite utilising OmniFit Benchmark Columns (Part Number – 006BCC-15-15-AF) - 15 x 150mm with one adjustable and one fixed end fitting. These columns have a column volume of ~18mL when packed to a bed height of 10cm. One column was packed with OmniSep A- Octyl Dash and the other with Octyl Sepharose FF. When the packing process was completed the columns were run sequentially on an Akta using identical conditions: Separation and Purification of α Chymotrypsinogen and Cytochrome C to Compare OmniSep™ A-Octyl Dash and Octyl Sepharose™ FF Columns
Transcript of Separation and Purification of α Chymotrypsinogen and ... CHROM3… · When the packing process...
Application Note: CHROM303
Introduction
ChromatographyAnalysis
For the comparison of OmniSep A-Octyl Dash and Octyl Sepharose FF the sample purified was a mixture of Cytochrome C, Lysozyme and α-Chymotrypsinogen.
Cytochrome C proteins are found loosely associated with the inner membrane of the mitochondrion. Cytochrome C is a small (molecular weight about 12,000 Dalton’s), highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an essential component of the electron transport chain, where it carries one electron. It is capable of undergoing oxidation and reduction, but does not bind oxygen.
Cytochrome C is a highly conserved protein across many species, found in animals, plants, and many unicellular organisms. Its structure consists of a chain of 100 amino acids. The Cytochrome C molecule has been studied in evolutionary biology; its amino acid sequence is conserved in mammals differing by only a few residues. For example, the sequences of cytochrome c in humans are identical to that of chimpanzees (our closest relatives).
Lysozyme, also known as muramidase is a glycoside hydrolase enzyme that damages bacterial cell walls. Lysozyme is present in a number of secretions, such as tears, saliva, human milk, and mucus and large amounts of lysozyme can be found in hen egg white.
Chymotrypsinogen is a proteolytic enzyme and is a precursor of chymotrypsin a digestive enzyme. It is constructed of a single polypeptide chain consisting of 245 amino acid residues and is synthesized in the cells of the pancreas and stored. The cell when stimulated by either a hormonal signal or a nerve impulse empties the stored contents into a duct leading into the duodenum. Chymotrypsinogen must be inactive until it gets to the digestive tract. This prevents damage to the pancreas and other organs.
Two identical columns were used for the evaluation, separation and purification. They were packed in the OmniSep manufacturing suite utilising OmniFit Benchmark Columns (Part Number – 006BCC-15-15-AF) - 15 x 150mm with one adjustable and one fixed end fitting. These columns have a column volume of ~18mL when packed to a bed height of 10cm.
One column was packed with OmniSep A- Octyl Dash and the other with Octyl Sepharose FF.
When the packing process was completed the columns were run sequentially on an Akta using identical conditions:
Separation and Purification of α Chymotrypsinogen and Cytochrome C to Compare OmniSep™ A-Octyl Dash and Octyl Sepharose™ FF Columns
Separation Conditions
Eluent Buffer A: Sodium Phosphate buffer pH 7 (0.1M) containing Ammonium Sulphate (1.7M)
Eluent Buffer B: Sodium Phosphate buffer pH 7 (0.1M)
Detection: UV 280 nm
Flow rate: 5 mL/min
Protein standard: Cytochrome C, Lysozyme and α Chymotrypsinogen at 6 mg/mL of each of the proteins made up in 3 mL of starting buffer (18 mgs of each of the proteins in 3 mL).
Columns were equilibrated with Eluent Buffer A for 3 column volumes (CV), 12 minutes
Time %B0 004 024 100
Gradient:
0.2mL sample injected
Results
Data was collected on the Akta using the Akta Unicorn software version 5.0 and the chromatograms integrated to provide values for N – Column Efficiency in plates per metre, As – Column Asymmetry and Plate Height.
Chromatograms and chromatographic reports from the Akta are included below (Fig. 1 and Fig.2) and show no significant difference in the separation profiles between the OmniSep A-Octyl Dash and Octyl Sepharose FF columns, with similar retention times, efficiencies and asymmetries.
The OmniSep A-Octyl Dash column is a “plug and play” alternative for the Octyl Sepharose FF column.
1. Sample Volume and Type E: PROTEINS
2. Column: OMNISEP DASH OCTYL
3. Eluent A: PHOSPHATE (0.1M) pH 7 + AMMONIUM SULPHATE (1.7M)
4. Eluent B: PHOSPHTAE (0.1M) pH 7
5. Remarks: RUN 2
No. Peak Name Ret Width at Half Height
Plate Height Plates/Meter 10% Left 10% Right Asymmetry
MIN MIN CM N/m MIN MIIN
1. -0.03 5.27 - - 6.82 -0.02 0.00
2. 19.97 1.81 0.01475 6778 18.79 22.11 1.81
3. 26.80 2.25 0.01277 7832 25.36 29.15 1.63
4. 40.97 0.65 0.00046 218722 40.58 42.11 2.90
1. Sample Volume and Type E: PROTEINS
2. Column: SEPHAROSE OCTYL
3. Eluent A: PHOSPHATE(0.1M) pH 7 AMM SULFATE (1.7 M)
4. Eluent B: PHOSPHATE 0.1 M pH 7
5. Remarks: RUN 2
No. Peak Name Ret Width at Half Height
Plate Height Plates/Meter 10% Left 10% Right Asymmetry
MIN MIN CM N/m MIN MIIN
1. -12.25 0.02 - - 12.25 -12.21 12.00
2. -12.13 0.02 - - 12.13 -12.09 12.00
3. 3.48 1.42 0.29887 335 2.43 5.09 1.54
4. 17.31 2.28 0.03133 3192 15.75 18.60 0.83
5. 20.44 2.19 0.02074 4822 18.60 22.22 0.97
Diba QC OmniSep A Dash Application 15mm059:10_UV1_280nm Diba QC OmniSep A Dash Application 15mm059:10_UV1_280nm@01,BASEM
-20
0 20 40 60
mAU
0.0 10.0 20.0 30.0 40.0 min
-0.03
19.97
26.80
40.97
80
Diba QC OmniSep A Dash Application 15mm048:10_UV1_280nm Diba QC OmniSep A Dash Application 15mm048:10_UV1_280nm@01,BASEM
0
50
100mAU
0.0 5.0 10.0 15.0 20.0 25.0
3.48
17.31
20.44
Figure 2: UV chromatogram of Cytochrome C, Lysozyme and α Chymotrypsinogen samples on GE Sepharose Octyl column
Figure 1: UV chromatogram of Cytochrome C, Lysozyme and α Chymotrypsinogen samples on OmniSep Dash Octyl column
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