Site-Directed Electrostatic Measurements with a Thiol-Specific pH-Sensitive Nitroxide:  Differentiating Local p<i>K</i> and Polarity Effects by High-Field EPR

Smirnov, Alex I.; Ruuge, Andres E.; Резников, В. А.; Voinov, Maxim A.; Grigor'ev, I. A.

doi:10.1021/ja048801f

Cited by 42 publications

(43 citation statements)

References 20 publications

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“…An attachment of this label to the protein sulfhydryl groups allows the probing of the local electrostatics by EPR (45). The use of highfield EPR with pH-sensitive methanethio-sulfonate labels opens a new possibility for the investigation of site-directed spin-labeled proteins, as was demonstrated for the label R7 (X = SSO 2 CH 3 ) (68,93). Recently, an imidazolidine label R8 was synthesized which has a pK a value close to the physiological range (pK a = 5.7 for the label alone and pKa = 6.15 when bound to the glutathione) (87), therefore extending the arsenal of the pH-sensitive methanethiosulfonate labels.…”

Section: Methods Of Thiol Detectionmentioning

confidence: 99%

“…Thus, thiol-targeted labels R6-R8 (Fig. 2) provide a tool for probing local electrostatics of the biological macromolecules (45,46,68,93). Figure 3A demonstrates the pH dependence of the nitrogen hyperfine splitting of the imidazolidine methanethiosulfonate label R7 (X = SSO 2 CH 3 ).…”

Section: Methods Of Thiol Detectionmentioning

confidence: 99%

“…3B), therefore opening an additional possibility for using this disulfide label as a pH-sensitive one. Recently the imidazolidine methanethiosulfonate label R7 (X = SSO 2 CH 3 ) was used for site-directed electrostatic measurements (68,93). Note that the R 2 SSR 2 and R7 (X = SSO 2 CH 3 ) form identical adducts, R 2 SSB, in the reaction with thiol BSH.…”

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confidence: 99%

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Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Swartz

Khan

Khramtsov

2007

Antioxidants & Redox Signaling

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The aim of this article is to provide an overview of how electron paramagnetic resonance (EPR) can be used to measure redox-related parameters in vivo. The values of this approach include that the measurements are made under fully physiological conditions, and some of the measurements cannot be made by other means. Three complementary approaches are used with in vivo EPR: the rate of reduction or reactions of nitroxides, spin trapping of free radicals, and measurements of thiols. All three approaches already have produced unique and useful information. The measurement of the rate of decrease of nitroxides technically is the simplest, but difficult to interpret because the measured parameter, reduction in the intensity of the nitroxide signal, can occur by several different mechanisms. In vivo spin trapping can provide direct evidence for the occurrence of specific free radicals in vivo and reflect relative changes, but accurate absolute quantification remains challenging. The measurement of thiols in vivo also appears likely to be useful, but its development as an in vivo technique is at an early stage. It seems likely that the use of in vivo EPR to measure redox processes will become an increasingly utilized and valuable tool. OVERVIEWThe goal of this article is to indicate how the unique capabilities of EPR can be used very effectively to follow redox status/events in vivo. This will be done in the context of a general overview and two more detailed complementary mini-reviews of the current status of the use of in vivo EPR to measure free radicals and thiols. The article also seeks to delineate areas where developments are needed and the potential pathways to achieve them.The ability to measure redox status and redox processes in vivo is very attractive for several reasons. It has been clearly established that redox-active species are intrinsically involved in many physiological and pathophysiological processes. The roles of these species include being essential intermediates in key physiological reactions and, in many cases, being directly involved in the causal chain leading to pathology. The redox state is an important parameter for many key events in cell signaling (13). Also, therapeutic approaches for many types of pathophysiology involve redox reactions (84).Whereas many useful insights can be achieved by measurements in model systems and cell cultures, the complexity of redox processes and physiology makes it difficult to understand fully the redox processes that occur in vivo without making direct measurements with the intact NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript system. The dynamics of the vascular system, metabolism in different organs, and the complex web of oxidants and antioxidants, cannot be modeled readily. There are few techniques that are capable of making such measurements in vivo, but fortunately EPR can follow many of the most important aspects of these complex processes.Some of the redox-active species are free radicals, and for these, EPR is t...

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Section: Methods Of Thiol Detectionmentioning

confidence: 99%

Section: Methods Of Thiol Detectionmentioning

confidence: 99%

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Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Swartz

Khan

Khramtsov

2007

Antioxidants & Redox Signaling

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“…Also pH sensitive spin probes have been used to label the thiol group, e.g. of a synthetic peptide fragment of the laminin B1 chain (Smirnov et al, 2004).…”

Section: Spin Labeling Of Cysteinesmentioning

confidence: 99%

Site-Directed Spin Labeling and Electron Paramagnetic Resonance (EPR) Spectroscopy: A Versatile Tool to Study Protein-Protein Interactions

Klare¹

2012

Protein Interactions

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“…If nitroxides can be selectively targeted to metastatic cells in vivo, EPR imaging becomes an attractive modality to image micrometastatic lesions. This is because nitroxides can be imaged with high contrast at micromolar concentrations and can furthermore be chemically tailored to report cellular physiological information such as regional changes in pH (Halpern et al, 1989;Smirnov et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Clearance and Biodistribution of Liposomally Encapsulated Nitroxides: A Model for Targeted Delivery of Electron Paramagnetic Resonance Imaging Probes to Tumors

Burks¹,

Legenzov²,

Rosen³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Electron paramagnetic resonance (EPR) imaging using nitroxides as molecular probes is potentially a powerful tool for the detection and physiological characterization of micrometastatic lesions. Encapsulating nitroxides in anti-HER2 immunoliposomes at high concentrations to take advantage of the "self-quenching" phenomenon of nitroxides allows generation of robust EPR signals in HER2-overexpressing breast tumor cells with minimal background from indifferent tissues or circulating liposomes. We investigated the in vivo pharmacological properties of nitroxides encapsulated in sterically stabilized liposomes designed for long circulation times. We show that circulation times of nitroxides can be extended from hours to days; this increases the proportion of liposomes in circulation to enhance tumor targeting. Furthermore, nitroxides encapsulated in sterically stabilized anti-HER2 immunoliposomes can be delivered to HER2-overexpressing tumors at micromolar concentrations, which should be imageable by EPR. Lastly, after in vivo administration, liposomally encapsulated nitroxide signal also appears in the liver, spleen, and kidneys. Although these organs are spatially distinct and would not hinder tumor imaging in our model, understanding nitroxide signal retention in these organs is essential for further improvements in EPR imaging contrast between tumors and other tissues. These results lay the foundation to use liposomally delivered nitroxides and EPR imaging to visualize tumor cells in vivo.

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Site-Directed Electrostatic Measurements with a Thiol-Specific pH-Sensitive Nitroxide: Differentiating Local pK and Polarity Effects by High-Field EPR

Cited by 42 publications

References 20 publications

Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

Site-Directed Spin Labeling and Electron Paramagnetic Resonance (EPR) Spectroscopy: A Versatile Tool to Study Protein-Protein Interactions

Clearance and Biodistribution of Liposomally Encapsulated Nitroxides: A Model for Targeted Delivery of Electron Paramagnetic Resonance Imaging Probes to Tumors

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