What are Biological Buffers?
Different inorganic substances were originally used as buffers (e.g. phosphate, cacodylate, borate, bicarbonate), and later weak organic acids were also used. Many of these buffer substances, however, have the disadvantage that they are not inert and have lasting effects on the system under investigation (e.g. inhibition of enzymes, interactions with enzyme substrates etc.). Most biological buffers in use today were developed by NE Good and his research team (Good et al. 1966, Good & Izawa 1972, Ferguson et al. 1980; “Good buffers”) and are N-substituted taurine or glycine buffers. These zwitterionic buffers meet most of the requirements that biological buffers have to fulfil. Buffer systems described in the literature are usually used for experiments to enable direct comparison of results. Again and again, it is shown that the conditions in experiments – even in standard systems – could be optimised.
We have put together the information from the literature that we believe will be of assistance to you in solving your everyday problems and in the development and optimisation of your test systems. The brochure " Biological Buffers" imparts basic knowledge on biological buffer substances and support in terms of selecting the best buffer substance for your assay system. Be aware of the fact that some buffers serve as substrate / inhibitor of enzymes (e.g. phosphate) or the temperature sensitivity of the pH of some buffers, especially Tris, probably the most frequently employed buffer substance.
If an enzyme is to be investigated, the first step is usually to determine the conditions under which the enzyme will show the highest possible degree of stability and activity. The determination of the pH optimum is important as the initial step in this. The pKa value of a buffer, i.e. the mid-point of its pH range, should be as near as possible to the desired pH value for the buffer being used, in other words, it should correspond to the pH optimum of the enzyme under testing. An adequate buffer capacity is often only reached at concentrations higher than 25 mM. However, higher buffer concentrations and related high ionic strengths can inhibit enzyme activity. Suitable initial concentrations are therefore between 10 and 25 mM. More detailed information about Criteria for the selection of a buffer you will find in our brochure.
Requirements of biological buffers
• Solubility: The buffer should be freely soluble in water and poorly soluble in other solvents.
• Permeability: The buffer should not be able to permeate biological membranes to prevent concentration in the cell or organelles. Tris has a relatively high degree of fat solubility and may therefore permeate membranes. This also explains its toxicity for many mammalian cells in culture.
• Ionic strength: The buffer should not alter the ionic strength of the system as far as possible. The physiological ionic strength is between 100 – 200 mM KCl or NaCl.
• Dependence of the pKa value: The pKa value of a buffer should be influenced as little as possible by the buffer concentration, the temperature and the ion composition of the medium. Amongst the buffers with temperature dependent pKa values, for example, are the amine buffers, whilst carboxylic acid buffers generally react less sensitively to changes in temperature.
• Complex formation: When a buffer forms complexes with metal ions, protons are released, which causes the pH value to decrease.
•Inert substances: The buffer should not be subject to either enzymatic or non-enzymatic changes, i.e. it should not be an enzyme substrate or enzyme inhibitor and should not react with metabolites or other components.
•UV absorption: Buffers should not absorb any light at wave-lengths longer than 230 nm, since many spectrophotometric investigations are performed in this range.
• Purity: Buffers should be as easy to manufacture and purify as possible. Purity is extremely important, since contaminations (e.g. heavy metals) can easily interfere with sensitive biological systems.
• Costs: When purifying proteins, large amounts of buffer are often need for centrifugation, chromatography steps or dialysis. The costs for materials therefore affect the planning of an experiment.