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Bovine pancreatic deoxyribonuclease F: isoelectric focusing, peptide mapping and primary structure
1Institute of Biochemistry, National Taiwan University/College of Medicine, Taipei.
Abstract:
DNAase F is a minor isoform of bovine pancreatic DNAase which can be separated from the other isoforms (DNAases A, B, C and D) present in a commercial preparation by a preparative isoelectric-focusing cell (Rotofor; Bio-Rad). The ampholytes and other contaminating proteins present in DNAase F preparations can be removed by chromatography on an affinity column (Cibacron Blue 3GA-agarose) and a hydrophobic-interaction column (phenyl-Sepharose CL-4B). The complete separation of DNAase F from the other isoforms is demonstrated on a thin-layer isoelectric-focusing gel, DNAase F being the most basic (pI 5.68). A procedure is described for tryptic peptide mapping by h.p.l.c. requiring only picomolar amounts of DNAase F protein. The DNAase F map shows two peptide peaks not present in the DNAase A map, and the DNAase F map does not have a peak at the position where a C-terminal peptide of DNAase A is normally eluted. The amino acid compositions and sequences for the two new peptides suggest that Gly240 in DNAase A is changed to Arg240 in DNAase F.
Insights
Researchers isolated DNAase F, a minor bovine pancreatic deoxyribonuclease (DNAase) isoform, using isoelectric focusing. Peptide mapping revealed a key amino acid difference, suggesting Glycine at position 240 in DNAase A is replaced by Arginine in DNAase F.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Bovine pancreatic deoxyribonuclease (DNAase) exists as multiple isoforms.
- DNAase F is a minor isoform that requires specific isolation techniques.
Purpose of the Study:
- To isolate and characterize the minor DNAase F isoform from bovine pancreatic DNAase.
- To elucidate the structural differences between DNAase F and other DNAase isoforms, particularly DNAase A.
Main Methods:
- Preparative isoelectric focusing (Rotofor) for initial separation.
- Affinity chromatography (Cibacron Blue 3GA-agarose) and hydrophobic-interaction chromatography (phenyl-Sepharose CL-4B) for purification.
- Thin-layer isoelectric focusing for assessing separation completeness.
- High-performance liquid chromatography (HPLC) for tryptic peptide mapping.
Main Results:
- DNAase F was successfully separated from other DNAase isoforms (A, B, C, D) and purified.
- DNAase F exhibits the most basic isoelectric point (pI 5.68).
- Tryptic peptide mapping identified two unique peptides in DNAase F and the absence of a C-terminal peptide found in DNAase A.
- Amino acid analysis indicated a Glycine to Arginine substitution at position 240 (Gly240Arg240) in DNAase F compared to DNAase A.
Conclusions:
- A robust method was developed for isolating and purifying the minor DNAase F isoform.
- The study identified a specific amino acid substitution responsible for the distinct properties of DNAase F.
- This research contributes to the understanding of DNAase isoform diversity and structure-function relationships.