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  • The Internet Journal of Genomics and Proteomics
  • Volume 2
  • Number 2

Original Article

General Comments on Buffers

R Lundblad

Citation

R Lundblad. General Comments on Buffers. The Internet Journal of Genomics and Proteomics. 2006 Volume 2 Number 2.

Abstract
 

The major factor in biological pH control in eukaryotic cells is the carbon dioxoide-biocarbonate-carbonate buffer (Scheme I) system1,2,3,4. There other biological buffers such as bulk protein and phosphate anions which can provide some buffering effect, metabolites such as lactic acid which can lower pH and tris(hydroxylmethylaminomethyl) methane, THAM®) has been used to treat acid base disorders5,6,7. pH control in prokaryotic cells is mediated by membrane transport of various ions including hydrogen, potassium and sodium8,9,10.

Figure 1

In the laboratory, the bicarbonate/carbonate buffer system can only be used in the far alkaline range (pH 9-11) and unless “fixed” by a suitable cation such as sodium, can be volatile.

A variety of buffers, most notably the “Good” buffers which were developed by Norman Good and colleagues[[[10a]]], have been developed over the years to provide pH control in in vitro experiments. While effective in controlling pH , the numerous non-buffer effects that buffer salts have on experimental systems are somewhat less appreciated. Some effects, such as observed with phosphate buffers, are based on biologically significant interactions with proteins and, as such, demonstrate specificity. Other effects, such as metal ion chelation, can be considered general. However, the binding of metal ions by a specific buffer must be carefully evaluated considering the recent controversy regarding the ability of MOPS buffer to bind magnesium ions11. There are some effects where the stability of a reagent is dependent on both pH and buffer species. One example is provided by the stability of phenylmethylsulfonyl fluoride(PMSF)12. PMSF was less stable in Tris buffer than in either HEPES or phosphate buffer; PMSF is less stable in HEPES than in phosphate buffer. Activity was measured by the ability of PMSF to inhibit chymotrypsin; all activity was lost in Tris (10 mM; pH 7.5) after one hour at 25°C while activity was fully retained in phosphate (10 mM, pH 7.5). This is likely a reflection of the nucleophilic property of Tris13,14 which appears to be enhanced in the presence of divalent cations such as zinc15. The loss of activity, presumably the result of the hydrolysis of the fluoride to hydroxyl function, is more marked at more alkaline pH. Tris can also function as phosphoacceptor in assays for alkaline phosphatase but was not as effective as 2-amino-2-methyl-1,3-propanediol16. The various nitrogen-based buffers such as Tris, HEPES, CAP, and BICINE influence colorimetric protein assays 17,18,19.

Other specific examples are presented in Table 1.

Figure 2
Table 1: Effects of Buffers

References to Table 1

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2. Durham, A.C., A survey of readily available chelators for buffering calcium ion concentrations in physiological solutions, Cell Calcium 4, 33-46, 1983

3. Stellwagen, N.C., Bossi, A., Gelfi, C. and Righetti, P.G., DNA and buffers: Are there any noninteracting neutral pH buffers?, Anal.Biochem. 287, 167-175, 2000

4. Syvertsen, C. and McKinley-McKee, J.S., Affinity labelling of liver alcohol dehydrogenase. Effect of pH and buffers on affinity labelling with iodoacetic acid and (R,S)-2- bromo-3-(5-imidazolyl)propionic acid, Eur.J.Biochem. 117, 165-170, 1981

5. Biyani, M. and Nishigaki, K., Sequence-specific and nonspecific mobilities of single-stranded oligonucleotides observed by changing the borate buffer concentration, Electrophoresis 24, 628-633, 2003

6. Zittle, Z.A., Reaction of borate with substances of biological interest, Adv.Enzymol.Relat.Sub.Biochem. 12, 493-527, 1951

7. Weitzman, S., Scott, V., and Keegstra, K., Analysis of glycoproteins as borate complexes by polyacrylamide gel electrophoresis, Anal.Biochem. 438-449, 1979

8. Patthy, L. and Smith, E.L., Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues, J.Biol.Chem. 250, 557-564, 1975

9. Jacobson, K.B., Murphey, J.B., and Sarma, B.D., Reaction of cacodylic acid with organic thiols, FEBS Lett. 22, 80-82, 1972

10. Cheung, S.T. and Fonda, M.L., Reaction of phenylglyoxal with arginine. The effect of buffers and pH, Biochem.Biophys.Res.Commun. 90, 940-947, 1979

10a. Good, N.E., Winget, G.D., Winter, W., et al., Hydrogen ion buffers for biological research, Biochemistry 5, 467-477, 1966

11. Uppu, R.M., Squadrito, G.L., and Pryor, W.A., Acceleration of peroxynitrite oxidations by carbon dioxide, Arch.Biochem.Biophys. 327, 335-343, 1996

12. Denicola, A., Freeman, B.A., Trujillo, M., and Radi, R., Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations, Arch.Biochem.Biophys. 333, 49-58, 1996

13. Munday, R., Munday, C.M. and Winterbourn, C.C., Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of iron salts, Free Rad.Biol.Med. 36, 757-764, 2004

14. Jansson, P.J., Del Castillo, U., Lindqvist, C., and Nordstrom, T., Effects of iron on vitamin C/copper-induced hydroxyl radical generation in bicarbonate-rich water, Free Rad.Res. 39, 565-570, 2005

15. Ramirez, D.C., Mejiba, S.E. and Mason, R.P., Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells, J.Biol.Chem. 280, 27402-27411, 2005

16. Tadolini, B., Iron autoxidation in Mops and Hepes buffers, Free Radic.Res.Commun. 4, 149-160, 1987

17. Simpson, J.A., Cheeseman, K.H., Smith, S.E., and Dean, R.T., Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer, Biochem.J. 254, 519-523, 1988

18. Sokolowska, M. and Bal, W., Cu(II) complexation by "non-coordinating" N-2-hydroxyethylpiperazine-N'-enthanesulfonic acid (HEPES buffer), J.Inorgan.Biochem. 99, 1653-1660, 2005

19. Bowman, C.M., Berger, E.M., Butler, E.N. et al., HEPES may stimulate cultured endothelial-cells to make growth-retarding oxygen metabolites, In Vitro Cell.Devel.Biol. 21, 140-142, 1985

20. Magonet, E., Briffeuil, E., Polimay, Y., and Ronveaux, M.F., Adverse-effects of HEPES on human-endothelial cells in culture, Anticancer Res. 7, 901, 1987

21. Mash, H.E., Chin, Y.P., Sigg, L., et al., Complexation of copper by zwitterionic aminosulfonic (Good) buffers, Anal.Chem. 75, 671-677, 2003

22. Altura, B.M., Carella, A., and Altura, B.T., Adverse effects of Tris, HEPES, and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins, Prostaglandins Med. 5, 123-130, 1980

23. Tadolini, B., and Sechi, A.M., Iron oxidation in Mops and Hepes buffers, Free Radic.Res.Commun. 4, 149-160, 1987

24. Schmidt, K., Pfeiffer, S., and Meyer, B., Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors, Free Radic.Biol.Med. 24, 859-862, 1998

25. Zhao, G. and Chasteen, J.D., Oxidation of Good's buffers by hydrogen peroxide, Anal.Biochem. 349, 262-267, 2006

26. Dudley, K.H. and Bius, D.L., Buffer catalysis of the racemization reaction of some 5-phenylhydantoins and its relation to in vivo metabolism of ethotoin, Drug.Metab.Dispos. 4, 340-348, 1976

27. Lazarus, R.A., Chemical racemization of 5-benzylhydantoin, J.Org.Chem. 55, 4755-4757, 1990

28. Moore, S.A., Kingston, R.L., Loomes, K.M., et al., The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insight into heparin binding, J.Mol.Biol. 312, 511-523, 2001

29. Schmidt, J., Mangold, C., and Deitmer, J., Membrane responses evoked by organic buffers in identified leech neurones, J.Exp.Biol. 199, 327-335, 1996

30. Robinson, J.D. and Davis, R.L., Buffer, pH, and ionic strength effects on the (Na+ + K+)-ATPase, Biochim.Biophys.Acta 912, 343-347, 1987

31. Poole, C.A., Reilly, H.C., and Flint, M.H., The adverse effects of HEPES, TES, and BES zwitterionic buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes, In Vitro 18, 755-765, 1982

32. Pogány, G., Hernandez, D.J., and Vogel, K.G., The in Vitro interaction of proteoglycans with type I collagen is modulated by phosphate, Archs.Biochem.Biophys. 313, 102-111, 1994

33. Grande, H.J. and Van der Ploeg, K.R., Tricine radicals as formed in the presence of peroxide producing enzymes, FEBS Lett. 95, 352-356. 1978

34. Oliver, R.W. and Viswanatha, T., Reaction of tris(hydroxymethyl)aminomethane with cinnamoyl imidazole and cinnamoyltrypsin, Biochim.Biophys.Acta 156, 422-425, 1968

35. Ray, T., Mills, A., and Dyson, P., Tris-dependent oxidative DNA strand scission during electrophoresis, Electrophoresis 16, 888-894, 1995

36. Qi, Z., Li, X., Sun, D., et al., Effect of Tris on catalytic activity of MP-11, Bioelectrochemistry 68, 40-47, 2006

References

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11. Montigny, C. and Champeil, P., Use of metallochromatic dyes and potentiomeric pH-meter titration to detect binding of divalent cations to "Good's" buffers: 4-morpholinepropanesulfonic acid (Mops) does not bind Mg2+, Analyt.Biochem. 366, 96-98, 2007
12. James, G.T., Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers, Anal.Biochem. 86, 574-579, 1978
13. Acharya, A.S., Roy, R.P., and Dorai, B., Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin A: preferential inhibition of glycation by nucleophiles at sites of low isomerization potential, J.Protein Chem. 10, 345-358, 1991
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Author Information

Roger L. Lundblad

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