Preparation of heme-free soluble guanylate cyclase.
Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) 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 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees 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 1M 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.
Schmidt
Peter
P
Institute of Cardiovascular Research, Bayer AG, Aprather-Weg 18a, D-42096, Wuppertal, Germany.
Schramm
Matthias
M
Schröder
Henning
H
Stasch
Johannes Peter
JP
D001565:Benzoates / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000302:isolation & purification; D006420:Hemeproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007191:Indazoles / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D011136:Polysorbates / Q000737:chemistry; D011506:Proteins / Q000032:analysis; D011524:Protoporphyrins / Q000737:chemistry; D013053:Spectrophotometry; D015032:Zinc / Q000737:chemistry
D001565:Benzoates; D020536:Enzyme Activators; D006420:Hemeproteins; D007191:Indazoles; D011136:Polysorbates; D011506:Proteins; D011524:Protoporphyrins; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009599:Nitroprusside; C480588:BAY 58-2667; D006418:Heme; D006639:Histidine; D006160:Guanosine Triphosphate; C028025:protoporphyrin IX; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D015032:Zinc