[12 Lysozyme Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria

By Victor L. Davidson and Dapeng Sun

Introduction

The cell envelope of gram-negative bacteria is composed of a cytoplasmic membrane (plasma membrane), a murein peptidoglycan layer, and an outer membrane that is linked by lipoproteins to the murein layer. The compartment between the cytoplasmic and outer membrane is called the periplasm, or periplasmic space.1 Several proteins are specifically localized in the periplasm. These have long been known to include binding proteins for nutrients, and hydrolytic and degradative enzymes. More recently it has become clear that the periplasm is also home to a wide variety of redox enzymes and electron transfer

1 J. W. Costerton, J. M. Ingram, and K.-J. Cheng, Bacteriol. Rev. 39, 87 (1974).

subsequent addition of at —30°, circumvents complications by the remaining reductant, because at that temperature reduction of NOS by sodium dithionite is extremely slow (t\n on the order of weeks; our unpublished observations, 1998). It is this simple and effective way to achieve a situation in which only the electron on the heme is available for oxygen reduction that enabled us to conclude that BH4 must serve as the donor of the second electron in the reaction cycle with arginine. Furthermore, as the sample chamber is continuously flushed with nitrogen, anaerobic conditions are guaranteed. One can apply the same experimental procedure to generate samples for other spectroscopic techniques, such as electron paramagnetic resonance (EPR) spectroscopy.23

One disadvantage of the method is that about 2 min must elapse before the first spectrum after O2 addition can be measured. It is mainly because of this limitation that, for some intermediates, spectral decomposition, and hence determination of the absorption spectra, could not be achieved. This can and will be redressed by application of low-temperature stopped-flow spectroscopy in future experiments.

Acknowledgments

This work was supported by Grant 13013-MED of the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich and the Human Frontier Science Program (RGP 0026/2001-M).

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