Difference between revisions of "Cyanide"

Revision as of 12:30, 28 October 2020

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

Application in HRR

Inhibitor titrations, CIV-assay, alternative oxidase and ROX

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

Caution: KCN is photosensitive and hygroscopic; HCN 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).

Preparation of 45 mM Trizma base:

Weigh 545 mg of Trizma base
Dissolve it in 100 mL of H₂0.

Preparation of 20 mM KCN stock solution (in 45 mM Trizma base) for 2-mL O2k-chamber:

Weigh 5.2 mg of KCN and dissolve in 4 mL of 45 mM Trizma base.
Divide into 500 µL aliquots (use dark glass vials).
Store frozen at -20 °C.

» 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 0.25 M 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

@@ Line 16: / Line 16: @@
 :::: <span style="color:#8B008B"> '''Caution:''' KCN is photosensitive and hygroscopic; HCN 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 20 mM KCN stock (in 45 mM Trizma base)'''
 :::: '''Preparation of 45 mM Trizma base:'''
 ::::# Weigh 545 mg of Trizma base
 ::::# Dissolve it in 100 mL of H<sub>2</sub>0.
-:::: '''Preparation of 20 mM KCN:'''
+::::''' Preparation of 20 mM KCN stock solution ''' (in 45 mM Trizma base) for '''2-mL O2k-chamber''':
 ::::# Weigh 5.2 mg of KCN and dissolve in 4 mL of 45 mM Trizma base.
 ::::# Divide into 500 µL aliquots (use dark glass vials).
 ::::# Store frozen at -20 °C.
+:::» '''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 0.25 M 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: 100 µL of 20 mM stock (2 mL O2k-chamber).
+::::* Titration volume ('''0.5-mL O2k-chamber'''): 2 µL using a 10 µL Hamilton syringe.
 ::::* Final concentration: 1 mM.
 {{MitoPedia O2k and high-resolution respirometry|type=Inhibitor ETS