1. Choice of Buffer
The pKa of the buffer should be within 0.5 unit of
the desired pH (± 1 unit if you want to push it)
Potential interactions with a column matrix
Avoid UV-absorbing buffers if you plan to use a
UV detector
The ionic strength and salt composition must be
chosen according to the stability of the protein and
the detergent
2. In other words, you want a “Good” buffer:
Hydrogen Ion Buffers for Biological Research*
Norman E. Good, G. Douglas Winget, Wilhelmina Winter,
Thomas N. Connolly, Seikichi Izawa, and Raizada M. M.
Singh Biochemistry, 1966, 5 (2), 467-477• DOI:
7. Preparation of Buffers
How would one make 1 L of a 2.0 M stock solution of
Tris·Cl at pH 8.0?
8. How would one make 1 L of a 1.0 M stock solution of
K+·MES at pH 6.5?
9. Many enzymes require a particular metal ion for optimal
activity, and many of these enzymes are inhibited by
metal ions other than their physiological, activating ones.
What treatment should be done to a buffer solution to
ensure that the buffer is free of contaminating metal ions?
10. Let us say that you want to do a pH profile of an enzyme
(i.e., assay the log(activity) of the enzyme as a function of
pH. What precautions should you take before you engage
in such a study?
11. Let us say that the product of the enzyme-catalyzed
reaction that you are studying as a function of pH absorbs
at a particular wavelength, say 240 nm, but the substrate
does not absorb appreciably at this wavelength. Are
there any concerns about the lmax of the chromophore
product as the pH changes?
12. Let us say that the molar extinction coefficient of the
chromophore product does change with pH. It would be
nice if one could derive a relationship between the
molar extinction coefficient and pH (i.e., how the extinction
coefficient changes as a function of pH). Hint: we did a
similar derivation when we developed the theory behind
the “pKa of Indicator” lab. OK, so give it a try...
