Preparation of heme-free soluble guanylate cyclase

doi:10.1016/S1046-5928(03)00142-6

Protein Expression and Purification

Volume 31, Issue 1, September 2003, Pages 42-46

https://doi.org/10.1016/S1046-5928(03)00142-6 Get rights and content

Abstract

Soluble guanylate cyclase (sGC), a heterodimer consisting of α- and β-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the β-subunit via His₁₀₅ is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 μg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 °C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1 M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.

Section snippets

Materials

Centrifugal ion exchangers Vivapure QL were purchased from Vivascience (Vivascience, Hannover, Germany). The Bradford protein assay was obtained from Bio-Rad (Bio-Rad, München, Germany). YC-1 and BAY 58-2667 were synthesized, as described previously [12], [13]. Protoporphyrin IX zinc(II) complex (ZnPP) and sodium nitroprusside (SNP) were from Aldrich (Aldrich, München, Germany). All other chemicals of analytical grade were obtained from Sigma (Sigma, Taufkirchen, Germany).

Spectroscopic studies

UV/Vis spectra were

Results and discussion

This study was performed to establish a technique that allows easy and reliable separation of sGC from Tween 20 and unbound heme for routine processing of a large number of samples. The removal of the sGC heme moiety by incubation with the detergent Tween 20 was previously reported [11]. However, until now the subsequent separation of sGC from detergent and unbound heme has posed an unresolved problem.

For removal of the heme group, sGC was incubated with 2% Tween 20. Here, we demonstrate that

Conclusion

The ability to modulate the heme moiety of sGC is an important prerequisite for many investigations aimed at exploring basic mechanisms of sGC activation. Here, we present a simple and reliable method for the separation of sGC, detergent, and porphyrin, based on commercially available centrifugal ion exchangers. This approach results in an excellent purification of sGC and avoids any dilution of the enzyme. Moreover using this technique, it is possible to recover more than 70% of the applied

References (17)

L. Serfass et al.
Effect of heme oxygenase inhibitors on soluble guanylyl cyclase activity
Arch. Biochem. Biophys.
(1998)
A. Hobbs
Soluble guanylate cyclase
Emerging Ther. Targets
(2000)
C.C. Wu et al.
YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase
Br. J. Pharmacol.
(1995)
A. Friebe et al.
Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme
EMBO J.
(1996)
J.W. Denninger et al.
Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies
Biochemistry
(2000)
J.P. Stasch et al.
NO-independent regulatory site on soluble guanylate cyclase
Nature
(2001)
J.P. Stasch et al.
Cardiovascular actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies
Br. J. Pharmacol.
(2002)
J.P. Stasch et al.
NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle
Br. J. Pharmacol.
(2002)

There are more references available in the full text version of this article.

Cited by (17)

Maturation, inactivation, and recovery mechanisms of soluble guanylyl cyclase
2021, Journal of Biological Chemistry
Citation Excerpt :
Indeed, the only heme-free sGC component that is definitively present in mammalian cells is the immature apo-sGCβ subunit in complex with Hsp90 (53, 76). While purified sGC heterodimer can be rendered heme-free by mild detergent treatment (97, 98), the existence of heme-free heterodimer in cells and tissues has not been widely demonstrated. A notable exception occurs when pharmacological agonists such as BAY 58 or BAY 60 are added to cells that express heme-oxidized or apo-sGCβ along with sGCα.
Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric enzyme that generates many molecules of cGMP in response to its ligand nitric oxide (NO); sGC thereby acts as an amplifier in NO-driven biological signaling cascades. Because sGC helps regulate the cardiovascular, neuronal, and gastrointestinal systems through its cGMP production, boosting sGC activity and preventing or reversing sGC inactivation are important therapeutic and pharmacologic goals. Work over the last two decades is uncovering the processes by which sGC matures to become functional, how sGC is inactivated, and how sGC is rescued from damage. A diverse group of small molecules and proteins have been implicated in these processes, including NO itself, reactive oxygen species, cellular heme, cell chaperone Hsp90, and various redox enzymes as well as pharmacologic sGC agonists. This review highlights their participation and provides an update on the processes that enable sGC maturation, drive its inactivation, or assist in its recovery in various settings within the cell, in hopes of reaching a better understanding of how sGC function is regulated in health and disease.
A novel soluble guanylyl cyclase activator, BR 11257, acts as a non-stabilising partial agonist of sGC
2019, Biochemical Pharmacology
Citation Excerpt :
In contrast, activity induced by the sGC stimulator BAY 41-2272 was reduced after preincubation with ODQ (Fig. 1C). Since Tween 20 was shown to efficiently remove the heme moiety of sGC [42,43], activity measurements were performed in the presence and absence of 0.5% Tween 20 to support our findings. Fig. 1D shows that both the BR 11257 and the BAY 60-2770-induced activities were significantly increased in the presence of 0.5% Tween 20.
The soluble guanylyl cyclase (sGC) plays a key role in NO/cGMP signalling and is widely recognised to be important in different disease pathomechanisms. The discovery of sGC agonists provides a new opportunity to stimulate the NO/cGMP pathway. One class of compounds are the heme-independent sGC activators, which are thought to bind to oxidised or heme-free sGC. This enzyme is preferentially formed under disease situations accompanied by oxidative stress. Accordingly, this binding mode of sGC activators has quite some appeal for the clinical use of sGC activator drugs in diseases with high oxidative stress burden. However, none of the previous sGC activators, most of them dicarboxylic acid derivatives, has passed clinical trials to date, also because of the potent blood pressure lowering effects. In the current study, we investigate the effects of a new monocarboxylic drug BR 11257 in vitro and in vivo. Activity measurements with purified enzyme indicated gentle sGC activation for BR 11257 resembling a partial agonistic behaviour. In thermal shift measurements, we observed an unexpected difference between BR 11257 and the sGC activators from the dicarboxylic acid type. While activators from the dicarboxylic acid type had a highly thermostabilising influence on sGC, this effect was absent with BR 11257. We hypothesize that the key interaction partner for thermostabilisation is the second carboxylic acid in BAY 60-2770 which is missing in BR 11257. The absence of this thermodynamic receptor stabilisation and the partial agonism may be advantageous to overcome limitations of this class of drugs by avoiding excessive hypotension.
BAY 60–2770 activates two isoforms of nitric oxide sensitive guanylyl cyclase: Evidence for stable insertion of activator drugs
2018, Biochemical Pharmacology
Nitric oxide sensitive guanylyl cyclase (NOsGC), a hemoprotein and the major physiological receptor for nitric oxide (NO), is a heterodimer with the α₁/β₁ and α₂/β₁ isoforms known to be important for NO-signaling and conversion of GTP to cGMP in humans. Two innovative classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent sGC stimulators, that stimulate NOsGC directly and also increase the affinity towards NO, and the heme-independent sGC activators, that are thought to bind to oxidized and heme-free NOsGC in tissues exposed to oxidative stress. In the current study, we evaluate the effects of the sGC activators BAY 58–2667 (cinaciguat) and BAY 60–2770 on the isoforms α₁/β₁ and α₂/β₁ expressed in Sf9 cells. Western blot analysis of cytosolic fractions revealed a decrease in overexpressed NOsGC in the presence of sGC activators, which is dependent on an intact catalytic site of the enzyme. For both isoforms, we show a higher efficacy for BAY 60–2770 compared to cinaciguat after purification of NOsGC by affinity and size exclusion chromatography. Using a new experimental strategy of expression of NOsGC with activator and subsequent purification, we demonstrate a stable insertion of activator drugs into the enzyme during protein biosynthesis independent of the heme redox state. We postulate that the balance between stable insertion of activator during de novo synthesis and replacement of NOsGC ferric heme in tissues exposed to oxidative stress can be influenced by the dosage regimen.
The effects of nitroxyl (HNO) on soluble guanylate cyclase activity. Interactions at ferrous heme and cysteine thiols
2009, Journal of Biological Chemistry
Citation Excerpt :
Because both heme coordination (by NO) and cysteine thiol redox processes are known to be capable of regulating sGC activity (6, 37) it is clearly possible that the observed HNO-mediated changes in sGC activity could be due to either interaction or a combination of interactions at these two targets. Although removal of heme from sGC does not substantially alter basal activity (26), it drastically affected activation by both DEA/NO and HNO donors (Fig. 4A). Heme removal from sGC decreased its sensitivity to activation by DEA/NO (from 70-fold activation down to 20-fold) and markedly reduced the HNO-mediated activation.
It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.
Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase
2004, Journal of Biological Chemistry
Citation Excerpt :
Before the UV-visible spectra were recorded, the enzyme was separated by ion exchange chromatography from BAY 58-2667, ODQ, or free heme that might interfere with the measurement. Separation of sGC from Detergent—Separation of sGC from detergent and unbound heme was performed as previously described (26). Briefly, sGC-containing samples were loaded onto ion exchange columns and washed once to remove any traces of detergent.
Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the β₁-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.
NO-ferroheme is a signaling entity in the vasculature
2023, Nature Chemical Biology

View all citing articles on Scopus

View full text

Preparation of heme-free soluble guanylate cyclase

Abstract

Section snippets

Materials

Spectroscopic studies

Results and discussion

Conclusion

Arch. Biochem. Biophys.

Soluble guanylate cyclase

Emerging Ther. Targets

YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase

Br. J. Pharmacol.

Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme

EMBO J.

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies

Biochemistry

NO-independent regulatory site on soluble guanylate cyclase

Nature

Cardiovascular actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies

Br. J. Pharmacol.

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Br. J. Pharmacol.