Dataset Information

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

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.

SUBMITTER: Williams-Smith DL

PROVIDER: S-EPMC1183705 | biostudies-other | 1977 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:IR spectroscopy has been widely applied in the study of photo-activated biological processes such as photosynthesis, but has not been applied to the study of reacting systems which require rapid mixing of aqueous solutions. This has been due in part to the high pressure needed to make an aqueous solution flow rapidly through the 50 microns optical pathlength between the plates in an IR cuvette suitable for use with 2H2O and the high viscosity of the concentrated protein solutions required to generate measurable IR signals. An apparatus, based largely on conventional stopped-flow technology, is described which achieves mixing well within the time-resolved performance (approximately 40 ms) of modern Fourier-transform IR (FTIR) spectrometers, since the dead time of the mixing device is approximately 15 ms. It has proved possible to achieve efficient mixing by using a simple six-jet mixing device. This is probably at least in part because of the high back pressure which develops when aqueous fluid is passed rapidly through the short pathlength of the cuvette and which promotes turbulent flow. Several examples of measurements of the deacylation of acylchymotrypsins are provided which demonstrate the operation of the apparatus in conjunction with a spectrometer capable of scanning at four scans/s. For cinnamoyl-chymotrypsin, isotope-edited spectra have been obtained which show somewhat lower resolution than is achieved by conventional scanning methods, since some smoothing has to be applied to the spectra. Difference spectra of the acylation of chymotrypsin by glycylglycine p-nitrophenyl ester have been obtained by averaging ten stopped-flow shots and show good signal-to-noise ratio without smoothing. It is predicted that this apparatus is likely to find a variety of applications in the study of enzyme-catalysed reactions, since the spectra are relatively rich in structural information, and isotope editing greatly enhances the interpretability of the spectra.

Project description:We present the analysis of formaldehyde (HCHO) in anhydrous methanol (CH3OH) as a case study to quantify HCHO in non-aqueous samples. At higher concentrations (C > 0.07 M), we detect a product of HCHO, methoxy methanol (MM, CH3OCH2OH), by Fourier transform infrared spectroscopy, FTIR. Formaldehyde reacts with CH3OH, CD3OH, and CD3OD as shown by FTIR with a characteristic spectral feature around 1,195 cm-1 for CH3OH used for the qualitative detection of MM, a formaldehyde derivative in neat methanol. Ab initio calculations support this assignment. The extinction coefficient for 1,195 cm-1 is in the order of 1.4 × 102 M-1cm-1, which makes the detection limit by FTIR in the order of 0.07 M. For lower concentrations, we performed the quantitative analysis of non-aqueous samples by derivatization with dinitrophenylhydrazine (DNPH). The derivatization uses an aqueous H2SO4 solution to yield the formaldehyde derivatized hydrazone. Ba(OH)2 removes sulfate ions from the derivatized samples and a final extraction with isobutyl acetate to yield a 1:1 methanol: isobutyl acetate solvent for injection for electrospray ionization (ESI). The ESI analysis gave a linear calibration curve for concentrations from 10 to 200 µM with a time-of-flight analyzer (TOF). The detection and quantification limits are 7.8 and 26 μM, respectively, for a linear correlation with R 2 > 0.99. We propose that the formaldehyde in CH3OH is in equilibrium with the MM species, without evidence of HCHO in solution. In the presence of water, the peaks for MM become less resolved, as expected from the well-known equilibria of HCHO that favors the formation of methylene glycol and polymeric species. Our results show that HCHO, in methanol does not exist in the aldehyde form as the main chemical species. Still, HCHO is in equilibrium between the production of MM and the formation of hydrated species in the presence of water. We demonstrate the ESI-MS analysis of HCHO from a non-aqueous TiO2 suspension in methanol. Detection of HCHO after illumination of the colloid indicates that methanol photooxidation yields formaldehyde in equilibrium with the solvent.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets