Buffers Lecture
12
The pK a 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 Choice of Buffer
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Buffers Lecture
Transcript of Buffers Lecture
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
Choice of 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:
In other words, you want a “Good” buffer:
Biochemistry, 1966, 5 (2), 467-477
Biochemistry, 1966, 5 (2), 467-477
Biochemistry, 1966, 5 (2), 467-477
Biochemistry, 1966, 5 (2), 467-477
Preparation of Buffers
How would one make 1 L of a 2.0 M stock solution ofTris·Cl at pH 8.0?
How would one make 1 L of a 1.0 M stock solution ofK+·MES at pH 6.5?
Many enzymes require a particular metal ion for optimalactivity, 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 toensure that the buffer is free of contaminating metal ions?
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 ofpH. What precautions should you take before you engagein such a study?
Let us say that the product of the enzyme-catalyzed reaction that you are studying as a function of pH absorbsat a particular wavelength, say 240 nm, but the substratedoes not absorb appreciably at this wavelength. Are there any concerns about the λmax of the chromophoreproduct as the pH changes?
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 extinctioncoefficient 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...
