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. 1977 Dec 1;167(3):593–600. doi: 10.1042/bj1670593

Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy.

D L Williams-Smith, R C Bray, M J Barber, A D Tsopanakis, S P Vincent
PMCID: PMC1183705  PMID: 23760

Abstract

Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BRAY R. C. Sudden freezing as a technique for the study of rapid reactions. Biochem J. 1961 Oct;81:189–193. doi: 10.1042/bj0810189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bray R. C., Vincent S. P., Lowe D. J., Clegg R. A., Garland P. B. Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12. Biochem J. 1976 Apr 1;155(1):201–203. doi: 10.1042/bj1550201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CHANCE B. The effect of pH upon the equilibria of catalase compounds. J Biol Chem. 1952 Feb;194(2):483–496. [PubMed] [Google Scholar]
  4. Cammack R., Barber M. J., Bray R. C. Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase. Biochem J. 1976 Aug 1;157(2):469–478. doi: 10.1042/bj1570469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clegg R. A. Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12. Biochem J. 1976 Mar 1;153(3):533–541. doi: 10.1042/bj1530533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Douzou P. The use of subzero temperatures in biochemistry: slow reactions. Methods Biochem Anal. 1974;22:401–512. doi: 10.1002/9780470110423.ch9. [DOI] [PubMed] [Google Scholar]
  7. Good N. E., Winget G. D., Winter W., Connolly T. N., Izawa S., Singh R. M. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467–477. doi: 10.1021/bi00866a011. [DOI] [PubMed] [Google Scholar]
  8. Hui-Bon-Hoa G., Douzou P. Ionic strength and protonic activity of supercooled solutions used in experiments with enzyme systems. J Biol Chem. 1973 Jul 10;248(13):4649–4654. [PubMed] [Google Scholar]
  9. Lowe D. J., Lynden-Bell R. M., Bray R. C. Spin-spin interaction between molybdenum and one of the iron-sulphur systems of xanthine oxidase and its relevance to the enzymic mechanism. Biochem J. 1972 Nov;130(1):239–249. doi: 10.1042/bj1300239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maurel P., Hoa G. H., Douzou P. The pH dependence of the hydrolysis of benzoyl-L-arginine ethyl ester in cooled mixed solvents. J Biol Chem. 1975 Feb 25;250(4):1376–1382. [PubMed] [Google Scholar]
  11. Olson J. S., Ballow D. P., Palmer G., Massey V. The reaction of xanthine oxidase with molecular oxygen. J Biol Chem. 1974 Jul 25;249(14):4350–4362. [PubMed] [Google Scholar]
  12. SAHA A., CAMPBELL D. H., SCHROEDER W. A. IMMUNOCHEMICAL STUDIES ON LIVER AND ERYTHROCYTE CATALASES FROM CATTLE, HORSE, RABBIT AND HUMAN. Biochim Biophys Acta. 1964 Apr 6;85:38–49. doi: 10.1016/0926-6569(64)90165-8. [DOI] [PubMed] [Google Scholar]
  13. Stirpe F., Della Corte E. The regulation of rat liver xanthine oxidase. Conversion in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type O). J Biol Chem. 1969 Jul 25;244(14):3855–3863. [PubMed] [Google Scholar]
  14. Williams-Smith D. L., Patel K. Induced changes in the electron paramagnetic resonance spectra of mammalian catalases. Biochim Biophys Acta. 1975 Oct 20;405(2):243–252. doi: 10.1016/0005-2795(75)90091-4. [DOI] [PubMed] [Google Scholar]

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