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Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis.

Evjenth R, Hole K, Ziegler M, Lillehaug JR - BMC Proc (2009)

Bottom Line: We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis.The method is highly reproducible and enables reliable quantification of both substrates and products.It is therefore well-suited to determine kinetic parameters of acetyltransferases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. rune.evjenth@mbi.uib.no

ABSTRACT
Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases.

No MeSH data available.


MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides(215 nm). Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the Vmax/Km (oligopeptides). Error bars indicate S.D. Experiments are performed in triplicates.
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Figure 9: MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides(215 nm). Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the Vmax/Km (oligopeptides). Error bars indicate S.D. Experiments are performed in triplicates.

Mentions: Our results indicated that the HPLC system is a solid method to study peptide acetylation. Using the non-radioactive HPLC method to detect the amount of acetylated oligopeptides, we determined hNaa30p enzyme kinetic constants for some in vitro oligopeptide substrates. Different concentrations of 1MLGTG-RRR24 peptides (30–350 μM) were used with fixed concentration of acetyl CoA (300 μM) and 80 nM of purified MBP-hNaa30p. When using the absorption signal at 215 nm, representing acetylated oligopeptides, we calculated the Km oligopeptide to be 283 μM with Vmax of 3.3 pmol * min-1 * pmol hNaa30p-1 (Table 2). In the same run, the production of CoA was recorded at 260 nm and used to calculate the corresponding kinetic constants. Here we observed that the Michaelis Menten plot based on CoA production generated a dose dependent curve from which a significantly lower Vmax was obtained (Figure 6) compared to when Vmax was calculated based on the production of acetylated oligopeptides (Figure 7). Km oligopeptide based on CoA production was determined to be 3.1 μM with a Vmax of 8.7 pmol * min-1 * pmol hNaa30p-1 (Table 2). It is important to note that approximately eight pmoles CoASH (Figure 6) were produced per pmole acetylated oligopeptide (Figure 7). Since theoretically one mole CoASH should be generated per mole acetylated oligopeptide, a discrepancy in CoASH production relative to acetylated oligopeptide was apparent. Km for acetyl CoA were with non linear regression determined to be approximately 14 μM with a Vmax of 2.1 pmol * min-1 * pmol hNaa30p-1 (Figure 8). V/K for selected substrates was calculated (Figure 9) and the S.D., indicated by error bars, were determined based on three independent experiments. The data demonstrate that substrate selectivity of the enzyme can be readily detected by the HPLC method.


Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis.

Evjenth R, Hole K, Ziegler M, Lillehaug JR - BMC Proc (2009)

MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides(215 nm). Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the Vmax/Km (oligopeptides). Error bars indicate S.D. Experiments are performed in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 9: MBP-hNaa30p specificity constants (V/K) for selected oligopeptides based on detection of acetylated oligopeptides(215 nm). Purified MBP-hNaa30p (80 nM) was incubated with selected oligopeptides and acetyl CoA (300 μM) in acetylation buffer for 30 minutes at 37°C. The acetylation kinetics was analysed by reverse phase HPLC. V/K is the Vmax/Km (oligopeptides). Error bars indicate S.D. Experiments are performed in triplicates.
Mentions: Our results indicated that the HPLC system is a solid method to study peptide acetylation. Using the non-radioactive HPLC method to detect the amount of acetylated oligopeptides, we determined hNaa30p enzyme kinetic constants for some in vitro oligopeptide substrates. Different concentrations of 1MLGTG-RRR24 peptides (30–350 μM) were used with fixed concentration of acetyl CoA (300 μM) and 80 nM of purified MBP-hNaa30p. When using the absorption signal at 215 nm, representing acetylated oligopeptides, we calculated the Km oligopeptide to be 283 μM with Vmax of 3.3 pmol * min-1 * pmol hNaa30p-1 (Table 2). In the same run, the production of CoA was recorded at 260 nm and used to calculate the corresponding kinetic constants. Here we observed that the Michaelis Menten plot based on CoA production generated a dose dependent curve from which a significantly lower Vmax was obtained (Figure 6) compared to when Vmax was calculated based on the production of acetylated oligopeptides (Figure 7). Km oligopeptide based on CoA production was determined to be 3.1 μM with a Vmax of 8.7 pmol * min-1 * pmol hNaa30p-1 (Table 2). It is important to note that approximately eight pmoles CoASH (Figure 6) were produced per pmole acetylated oligopeptide (Figure 7). Since theoretically one mole CoASH should be generated per mole acetylated oligopeptide, a discrepancy in CoASH production relative to acetylated oligopeptide was apparent. Km for acetyl CoA were with non linear regression determined to be approximately 14 μM with a Vmax of 2.1 pmol * min-1 * pmol hNaa30p-1 (Figure 8). V/K for selected substrates was calculated (Figure 9) and the S.D., indicated by error bars, were determined based on three independent experiments. The data demonstrate that substrate selectivity of the enzyme can be readily detected by the HPLC method.

Bottom Line: We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis.The method is highly reproducible and enables reliable quantification of both substrates and products.It is therefore well-suited to determine kinetic parameters of acetyltransferases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. rune.evjenth@mbi.uib.no

ABSTRACT
Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases.

No MeSH data available.