Brumbaugh-Ackers method

The calculation of stability constants is based on the fact that measurement of total ligand concentration at any point within column allows a determination of the amount bound to protein, since the cross-sectional areas of Vq and V, can be known frcm Independent experiments (ref. 34). When M is totally excluded the free ligand concentration can be calculated frcm equation 

For illustrating the principle of the method, suppose we have a column saturated with a solution of M and L, and for simplicity suppose they have equal concentration, say 4 units per unit volume (Fig. 3). Suppose that also oC and / are equal, i.e. half units both (the volume Vg taken by the gel is omitted). The apparent concentration of M and 

ML within the gel bed is half of that in the solution above the bed and the concentration of free L with an E of one unit will be according to equation (6) 

Consequently the binding ratio r as calculated from equation (2) will be 

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Figure 3. Hypothetical column for the method of Brumbaugh and Ackers. Aj and A j repre-sent absorbances measured through equilibrated gel column (see Fig. 2). As shown, total ligand content is different in equal segments Aj (L 4) and At> (L = 3). For other details, see the text.

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