Difference between revisions of "Cyanide"

Revision as of 12:40, 22 January 2021

high-resolution terminology - matching measurements at high-resolution

Cyanide

Description

Cyanide (usually added as KCN) is a competitive inhibitor of cytochcrome c oxidase (CIV). Inhibition is reversed by pyruvate and high oxygen levels.

Abbreviation: KCN

Reference: MiPNet09.12

MitoPedia topics: Inhibitor

Application in HRR

Inhibitor titrations, CIV-assay, alternative oxidase and ROX

Cyanide and determination of residual oxygen consumption (ROX).
Complex IV assay
alternative oxidase

Caution: Due to a confusion, a change in the procedure for preparing the KCN was done in April 2019 that recommended to prepare 20 mM KCN stock and titrate 100 µl. This now have been corrected to our previous recommendation of 1 M KCN stock solutions

O2k manual titrations MiPNet09.12 O2k-Titrations

Titration volume: 2 µl of 1 M stock, using a 10 µl syringe (2 ml O2k-chamber).
Final concentration: 1 mM.

Preparation of 1 M KCN stock

Kcn(KCN): Fluka 60178, 100 g, store at R.T.; FW = 65.12.

Caution: KCN is photosensitive and hygroscopic; KCN is volatile and very toxic!

Preparation of 1 M KCN stock solution for 2-mL O2k-chamber

Weigh 13 mg of KCN.

Dissolve in 0.2 ml H₂O.
The pH of the solution may be very alkaline - adjust to physiological pH with HCl.

          :::» O2k manual titrations  MiPNet09.12 O2k-Titrations

Titration volume (2-mL O2k-chamber): 100 µL using a 100 µL Hamilton syringe.

Final concentration: 1 mM.

Preparation of 250 mM KCN stock solution (in 45 mM Trizma base) for 0.5-mL O2k-chamber:

Weigh 16,28 mg of KCN and dissolve in 1 mL of 45 mM Trizma base.
Divide into 200 µL aliquots (use dark glass vials).
Store frozen at -20 °C.

» O2k manual titrations MiPNet09.12 O2k-Titrations

Titration volume (0.5-mL O2k-chamber): 2 µL using a 10 µL Hamilton syringe.
Final concentration: 1 mM.

MitoPedia topics: Inhibitor

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 |info=[[MiPNet09.12]]
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+{{MitoPedia O2k and high-resolution respirometry|type=Inhibitor ETS
+}}
+{{MitoPedia topics
+|mitopedia topic=Inhibitor
+}}
 __TOC__
 == Application in [[HRR]] ==
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 ::::* [[alternative oxidase]]
-:::: '''Kcn'''(KCN): Fluka 60178, 100 g, store at R.T.; FW = 65.12; '''Trizma base''': Sigma T6066, 100 g, store at R.T.; FW = 121.14
+:::: <span style="color:#8B008B"> '''Caution:''' Due to a confusion, a change in the procedure for preparing the KCN was done in April 2019 that recommended to prepare 20 mM KCN stock and titrate 100 µl. This now have been corrected to our previous recommendation of 1 M KCN stock solutions </span>
+::: '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
+::::* Titration volume: 2 µl of 1 M stock, using a 10 µl syringe (2 ml O2k-chamber).
+::::* Final concentration: 1 mM.
-:::: <span style="color:#8B008B"> '''Caution:''' KCN is photosensitive and hygroscopic; KCN is volatile and very toxic! It is recommended to prepare fresh KCN solution although it can be safely stored at -20°C. To avoid KCN evaporation, it is recommended to use alkaline buffers (such as 45 mM Trizma base, pH >10).</span>
+=== Preparation of 1 M KCN stock ===
+:::: '''Kcn'''(KCN): Fluka 60178, 100 g, store at R.T.; FW = 65.12.
-:::: '''Preparation of 45 mM Trizma base:'''
+:::: <span style="color:#8B008B"> '''Caution:''' KCN is photosensitive and hygroscopic; KCN is volatile and very toxic! </span>
-::::# Weigh 545 mg of Trizma base
+:::: ''' Preparation of 1 M KCN stock solution ''' for '''2-mL O2k-chamber'''
-::::# Dissolve it in 100 mL of H<sub>2</sub>0.
+::::# Weigh 13 mg of KCN.
-::::''' Preparation of 20 mM KCN stock solution ''' (in 45 mM Trizma base) for '''2-mL O2k-chamber''':
+::::# Dissolve in 0.2 ml H<sub>2</sub>O.
-::::# Weigh 5.2 mg of KCN and dissolve in 4 mL of 45 mM Trizma base.
+::::# The pH of the solution may be very alkaline - adjust to physiological pH with HCl.
-::::# Divide into 500 µL aliquots (use dark glass vials).
+           :::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-::::# Store frozen at -20 °C.
+::::* Titration volume ('''2-mL O2k-chamber'''): 100 µL using a 100 µL Hamilton syringe.
-:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-::::* Titration volume ('''2-mL O2k-chamber'''): 100 µL using a 100 µL Hamilton syringe.
 ::::* Final concentration: 1 mM.
 :::: '''Preparation of 250 mM KCN stock solution''' (in 45 mM Trizma base) for '''0.5-mL O2k-chamber''':