Biology:Eadie–Hofstee diagram
In biochemistry, an Eadie–Hofstee plot (or Eadie–Hofstee diagram) is a graphical representation of the Michaelis–Menten equation in enzyme kinetics. It has been known by various different names, including Eadie plot, Hofstee plot and Augustinsson plot. Attribution to Woolf is often omitted, because although Haldane and Stern[1] credited Woolf with the underlying equation, it was just one of the three linear transformations of the Michaelis–Menten equation that they initially introduced. However, Haldane indicated latter that Woolf had indeed found the three linear forms:[2]
In 1932, Dr. Kurt Stern published a German translation of my book Enzymes, with numerous additions to the English text. On pp. 119–120, I described some graphical methods, stating that they were due to my friend Dr. Barnett Woolf. [...] Woolf pointed out that linear graphs are obtained when [math]\displaystyle{ v }[/math] is plotted against [math]\displaystyle{ v x^{-1} }[/math], [math]\displaystyle{ v^{-1} }[/math] against [math]\displaystyle{ x^{-1} }[/math], or [math]\displaystyle{ v^{-1}x }[/math] against [math]\displaystyle{ x }[/math], the first plot being most convenient unless inhibition is being studied.
Derivation of the equation for the plot
The simplest equation for the rate [math]\displaystyle{ v }[/math] of an enzyme-catalysed reaction as a function of the substrate concentration [math]\displaystyle{ a }[/math] is the Michaelis-Menten equation, which can be written as follows:
- [math]\displaystyle{ v = {{Va} \over {K_\mathrm{m} + a}} }[/math]
in which [math]\displaystyle{ V }[/math] is the rate at substrate saturation (when [math]\displaystyle{ a }[/math] approaches infinity, or limiting rate, and [math]\displaystyle{ K_\mathrm{m} }[/math] is the value of [math]\displaystyle{ a }[/math] at half-saturation, i.e. for [math]\displaystyle{ v = 0.5V }[/math], known as the Michaelis constant. Eadie[3] and Hofstee[4] independently transformed this into straight-line relationships, as follows: Taking reciprocals of both sides of the equation gives the equation underlying the Lineweaver–Burk plot:
- [math]\displaystyle{ {1 \over v} = {1 \over V} + {K_\mathrm{m} \over V} }[/math] · [math]\displaystyle{ {1 \over a} }[/math]
This can be rearranged to express a different straight-line relationship:
- [math]\displaystyle{ v = V - K_\mathrm{m} }[/math] · [math]\displaystyle{ {v \over a} }[/math]
which shows that a plot of [math]\displaystyle{ v }[/math] against [math]\displaystyle{ v/a }[/math] is a straight line with intercept [math]\displaystyle{ V }[/math] on the ordinate, and slope [math]\displaystyle{ -K_\mathrm{m} }[/math] (Hofstee plot). In the Eadie plot the axes are reversed, but the principle is the same. These plots are kinetic versions of the Scatchard plot used in ligand-binding experiments.
Attribution to Augustinsson
The plot is occasionally attributed to Augustinsson[5] and referred to the Woolf–Augustinsson–Hofstee plot[6][7][8] or simply the Augustinsson plot.[9] However, although Haldane, Woolf or Eadie were not explicitly cited when Augustinsson introduced the [math]\displaystyle{ v }[/math] versus [math]\displaystyle{ v/a }[/math] equation, both the work of Haldane[10] and of Eadie[3] are cited at other places of his work and are listed in his bibliography.[5]:169 and 171
Effect of experimental error
Experimental error is usually assumed to affect the rate [math]\displaystyle{ v }[/math] and not the substrate concentration [math]\displaystyle{ a }[/math], so [math]\displaystyle{ v }[/math] is the dependent variable.[11] As a result, both ordinate [math]\displaystyle{ v }[/math] and abscissa [math]\displaystyle{ v/a }[/math] are subject to experimental error, and so the deviations that occur due to error are not parallel with the ordinate axis but towards or away from the origin. As long as the plot is used for illustrating an analysis rather than for estimating the parameters, that matters very little. Regardless of these considerations various authors[12][13][14] have compared the suitability of the various plots for displaying and analysing data.
Use for estimating parameters
Like other straight-line forms of the Michaelis–Menten equation, the Eadie–Hofstee plot was used historically for rapid evaluation of the parameters [math]\displaystyle{ K_\mathrm{m} }[/math] and [math]\displaystyle{ V }[/math], but has been largely superseded by nonlinear regression methods that are significantly more accurate when properly weighted and no longer computationally inaccessible.
Making faults in experimental design visible
As the ordinate scale spans the entire range of theoretically possible [math]\displaystyle{ v }[/math] vales, from [math]\displaystyle{ 0 }[/math] to [math]\displaystyle{ V }[/math] one can see at a glance at an Eadie–Hofstee plot how well the experimental design fills the theoretical design space, and the plot makes it impossible to hide poor design. By contrast, the other well known straight-line plots make it easy to choose scales that suggest that the design is better than it is. Faulty design, as shown in the right-hand diagram, is common with experiments with a substrate that is not soluble enough or too expensive to use concentrations above [math]\displaystyle{ K_\mathrm{m} }[/math], and in this case [math]\displaystyle{ V/K_\mathrm{m} }[/math] cannot be estimated satisfactorily. The opposite case, with [math]\displaystyle{ a }[/math] values concentrated above [math]\displaystyle{ K_\mathrm{m} }[/math] (left-hand diagram) is less common but not unknown, as for example in a study of nitrate reductase.[15]
See also
- Michaelis–Menten equation
- Lineweaver–Burk plot
- Hanes plot
- Direct linear plot
Footnotes and references
- ↑ Allgemeine Chemie der Enzyme. Dresden and Leipzig: Steinkopff. 1932. pp. 119–120. OCLC 964209806.
- ↑ "Graphical Methods in Enzyme Chemistry" (in en). Nature 179 (4564): 832. 1957. doi:10.1038/179832b0. ISSN 1476-4687. Bibcode: 1957Natur.179R.832H.
- ↑ 3.0 3.1 "The Inhibition of Cholinesterase by Physostigmine and Prostigmine". Journal of Biological Chemistry 146: 85–93. 1942. doi:10.1016/S0021-9258(18)72452-6.
- ↑ "Non-inverted versus inverted plots in enzyme kinetics". Nature 184 (4695): 1296–1298. October 1959. doi:10.1038/1841296b0. PMID 14402470. Bibcode: 1959Natur.184.1296H.
- ↑ 5.0 5.1 "Cholinesterases: A study in comparative enzymology.". Acta Physiologica Scandinavica 15: Supp. 52. 1948. https://archive.org/details/in.ernet.dli.2015.50571/page/n1051/mode/2up.
- ↑ "Angiotensin-converting enzyme activity is reduced in brain microvessels of spontaneously hypertensive rats". Journal of Neurochemistry 42 (6): 1655–1658. June 1984. doi:10.1111/j.1471-4159.1984.tb12756.x. PMID 6327909.
- ↑ "Ontogenesis of taurocholate transport by rat ileal brush border membrane vesicles". The Journal of Clinical Investigation 75 (3): 869–873. March 1985. doi:10.1172/JCI111785. PMID 2579978.
- ↑ "Evidence for a high-affinity sodium-dependent D-glucose transport system in the kidney". The American Journal of Physiology 253 (1 Pt 2): F151–F157. July 1987. doi:10.1152/ajprenal.1987.253.1.F151. PMID 3605346.
- ↑ "Limitations of Augustinsson plots". Computer Applications in the Biosciences 8 (5): 475–479. October 1992. doi:10.1093/bioinformatics/8.5.475. PMID 1422881.
- ↑ Enzymes. London, New York: Longmans, Green, & Company. 1930. OCLC 615665842.
- ↑ This is likely to be true, at least approximately, though it is probably optimistic to think that [math]\displaystyle{ a }[/math] is known exactly.
- ↑ "A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations.". The Journal of Biological Chemistry 240 (2): 863–869. February 1965. doi:10.1016/S0021-9258(17)45254-9. PMID 14275146.
- ↑ "A comparison of seven methods for fitting the Michaelis-Menten equation". The Biochemical Journal 149 (3): 775–777. September 1975. doi:10.1042/bj1490775. PMID 1201002.
- ↑ Fundamentals of Enzyme Kinetics (4th ed.). Weinheim, Germany: Wiley-Blackwell. 27 February 2012. pp. 51–53. ISBN 978-3-527-33074-4.
- ↑ Buc, J.; Santini, C. L.; Blasco, F.; Giordani, R.; Cárdenas, M. L.; Chippaux, M.; Cornish-Bowden, A.; Giordano, G. (1995). "Kinetic studies of a soluble αβ complex of nitrate reductase A from Escherichia coli: Use of various αβ mutants with altered β subunits". Eur. J. Biochem. 234 (3): 766–772. doi:10.1111/j.1432-1033.1995.766_a.x.
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