Lineweaver-Burk double-reciprocal method

Transforming the Michaelis-Menten equation into a linear form facilitates the graphical analyses of the data. One of the more widely used methods for accomplishing this is the double reciprocal or Lineweaver-Burk plot. > 2 Inverting both sides of Eqn. 7.19 and substituting Eqn. 7.20 gives Eqn. 7.24, a Lineweaver-Burk double reciprocal, Eqn. 7.24. 

Lineweaver-Burk plot Method of analyzing kinetic data (growth rates of enzyme catalyzed reactions) in linear form using a double reciprocal plot of rate versus substrate concentration. 

In each of these cases, the double-reciprocal plots according to the Lineweaver-Burk method are linear. The appearance of these plots, and the parameters obtained from them, are developed in Problem 4-15. 

Repeat Question 11, but graph the data as a linear, double reciprocal plot in the spirit of the Lineweaver-Burk equation (see Chapter 4). Plot l/ATm vs. 1 /(N/nt) and perform a linear regression to determine the best-fit line (Equation 4.a). The x-intercept corresponds to the KD of the DNA-netropsin complex. The KD value from this method should be more accurate than the estimation in Question 11. 

These hyperbolic equations are analogous to the Michaelis-Menten equation. Nonlinear regression is preferable to the method proposed in the 1960s by Kitz and Wilson, which necessitates a double-reciprocal linear transformation of the data (analogous to a Lineweaver-Burk plot) that can bias the estimates of /clnact and A). 

Lineweaver—Burk Method.8 The Michaelis-Menten equation may be algebraically rearranged to Eq. 2.22, yielding a third linear plot, called the Line-weaver-Burk or double-reciprocal plot ... 

Another method to obtain estimates for Km and is the rearrangement of the Michaelis-Menten equation to a linear form. The estimation for the initial velocities, Vo, from progress curves is not a particularly reliable method. A better way to estimate Vn is by the integrated Michaelis-Menten equation (Cornish-Bowden, 1975). Nevertheless, the graphical methods are popular among enzymolo-gists. The three most common linear transformations of the Michaelis-Menten equation are the Lineweaver-Burk plot of 1/Vo vs. 1/[S] (sometimes called the double-reciprocal plot), the Eadie-Hofstee plot, i.e. v vs. vo/[S], and the Hanes plot, i.e., [SJ/vo vs. [S] (Fig. 9.3). 

Despite its disadvantages, the double reciprocal plot of Lineweaver-Burk has its merits and is frequently used. It is the only one in which independent and dependent variables are separated. In fact, the Hanes plot contains the independent variable in the Y (dependent) axis, while the Eaddie-Hofstee plot contains the dependent variable in the X (independent) axis. The fact that variables are not separated in these two methods obscure to some extent the interpretation of the data, being the double reciprocal plot quite illustrative to determine kinetic mechanisms, as will be seen in the next section. 

For the estimation of and V ,axj different graphical plots can be obtained after measurement of the reaction rates over a wide range of substrate concentration. These methods are illustrated in Figure 6. The Lineweaver-Burk plot or double reciprocal... 

To clarify the mode of inhibition, the reaction was analyzed by the method of Lineweaver and Burk (1934) at two concentrations of aldehyde reagents and five concentrations of cytochrome c. The data were plotted by the double reciprocal procedure and the inhibition was found to be both competitive and noncompetitive in type. 

---