2,695
edits
Lidstrom:Buffers: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
no edit summary
No edit summary |
|||
| Line 4: | Line 4: | ||
pH = pK<sub>a</sub> + log[A<sup>-</sup>]/[HA] | pH = pK<sub>a</sub> + log[A<sup>-</sup>]/[HA] | ||
Buffers are usually used within | Buffers are usually used within pK ± 1. | ||
(source: [http://www.amazon.com/Enzyme-Assays-Practical-Approach-Series/dp/0199631425/ref=sr_1_1?ie=UTF8&qid=1383921457&sr=8-1&keywords=0199631425 ISBN 0-19-963142-5] pg 318) | (source: [http://www.amazon.com/Enzyme-Assays-Practical-Approach-Series/dp/0199631425/ref=sr_1_1?ie=UTF8&qid=1383921457&sr=8-1&keywords=0199631425 ISBN 0-19-963142-5] pg 318) | ||
== How to chose a buffer for enzyme assays== | |||
Information from [http://www.amazon.com/Enzyme-Assays-Practical-Approach-Series/dp/0199631425/ref=sr_1_1?ie=UTF8&qid=1383921457&sr=8-1&keywords=0199631425 ISBN 0-19-963142-5] pg 321. | |||
1) '''Chose the pH range you want.''' Use a buffer that is within pK± 1 because the buffering capacity will be low outside that range. The range that is used may be varied if it is known that a buffer has only to counter the effects of acid or base but not both. (Is only H+ produced?) The edge of a working range can be more satisfactorily used if the change in pH is towards the pK. | |||
2) Stability of the buffer, whether it interacts with the substrates, cofactors, or metal ions, the temperature coefficients of its pK, the ionic strength at which it is used, its absorbance in the UV region of the spectrum, its cost, and its availability free from contaminants. | |||
Most of the newer zwitterionic buffers do not appreciably bind divalent metal ions, are chemically stable, do not appreciably absorb light at wavelengths longer than 240nm, and can be made up as a concentrated stock solutions. | |||
Many of the buffers which have been in longer use have one or more disadvantages. They are generally cheaper, and if required in large quantities, e.g. for dialysis or column chromatography, may be used provided they have been tested or are known not to affect the enzyme in question. '''The following drawbacks associated with certain buffers should be borne in mind.''' | |||
* '''Phosphate''' | |||
** Phosphate buffers tend to precipitate Mg<sup>2+</sup>, Ca<sup>2+</sup>, Fe<sup>3+</sup>, and other polyvalent cations. | |||
** Phosphate being an important metabolite is known to inhibit a number of enzymes; e.g. kinases, dehydrogenases, carboxypeptidase, fumarase, urease, aryl sulphatase, adenoside deaminase, phosphoglucomutase, and other enzymes involving phosphate esters. | |||
** On the other hand, it may stabilize enzymes, e.g. phosphoribosepyrophosphate synthase. | |||
* '''Pyrophosphate"" | |||
** Pyroph | |||
== Resources== | == Resources== | ||
