PMID- 12963339
OWN - NLM
STAT- MEDLINE
DCOM- 20040609
LR  - 20191107
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 31
IP  - 1
DP  - 2003 Sep
TI  - Preparation of heme-free soluble guanylate cyclase.
PG  - 42-6
AB  - 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.
FAU - Schmidt, Peter
AU  - Schmidt P
AD  - Institute of Cardiovascular Research, Bayer AG, Aprather-Weg 18a, D-42096, 
      Wuppertal, Germany.
FAU - Schramm, Matthias
AU  - Schramm M
FAU - Schroder, Henning
AU  - Schroder H
FAU - Stasch, Johannes Peter
AU  - Stasch JP
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Benzoates)
RN  - 0 (Enzyme Activators)
RN  - 0 (Hemeproteins)
RN  - 0 (Indazoles)
RN  - 0 (Polysorbates)
RN  - 0 (Proteins)
RN  - 0 (Protoporphyrins)
RN  - 154453-18-6 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole)
RN  - 169D1260KM (Nitroprusside)
RN  - 329773-35-5 (BAY 58-2667)
RN  - 42VZT0U6YR (Heme)
RN  - 4QD397987E (Histidine)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - C2K325S808 (protoporphyrin IX)
RN  - EC 4.6.1.2 (Guanylate Cyclase)
RN  - H2D2X058MU (Cyclic GMP)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Benzoates/pharmacology
MH  - Chromatography, Ion Exchange
MH  - Cyclic GMP/metabolism
MH  - Enzyme Activation/drug effects
MH  - Enzyme Activators/pharmacology
MH  - Guanosine Triphosphate/metabolism
MH  - Guanylate Cyclase/chemistry/*isolation & purification/metabolism
MH  - Heme/*chemistry/isolation & purification
MH  - Hemeproteins/chemistry/drug effects/metabolism
MH  - Histidine/chemistry
MH  - Indazoles/pharmacology
MH  - Nitroprusside/pharmacology
MH  - Polysorbates/chemistry
MH  - Proteins/analysis
MH  - Protoporphyrins/chemistry
MH  - Spectrophotometry
MH  - Zinc/chemistry
EDAT- 2003/09/10 05:00
MHDA- 2004/06/21 10:00
CRDT- 2003/09/10 05:00
PHST- 2003/09/10 05:00 [pubmed]
PHST- 2004/06/21 10:00 [medline]
PHST- 2003/09/10 05:00 [entrez]
AID - S1046592803001426 [pii]
AID - 10.1016/s1046-5928(03)00142-6 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2003 Sep;31(1):42-6. doi: 10.1016/s1046-5928(03)00142-6.