APPENDIX
Method for Phosphorylcreatine Analysis
The method of Ennor and Stocken (25) was used to
determine phosphorylcreatine content of muscle samples.
This method determines creatine content before and after
a period of acid hydrolysis of phosphorylcreatine. The
key reaction for this determination is creatine with
diacetyl which yields a pink colored compound. The
intensity of the pink color is proportional to creatine
concentration when diacetyl is available in excess. The
optical density is determined with a spectrophotometer
at 525 nm.
Chemicals and Reagents for Deproteinization and Determination
I Perchloric Acid, stock 70-72% (Baker)
a) .8 M in 40% ethanol
b) .6 M (no ethanol)
II Potassium Carbonate, (Baker) 3 M containing
.5 M triethanolamine (Baker)
III Sodium Hydroxide (Malinkrodt)
a) 1 N
b) 4 N
IV Hydrochloric acid (Fisher)
a) IN
b) 4 N
V p Chloromercuric acid (Sigma) (also called p
Hydroxymercuric acid) .05 M purchased as
solution, or as powder then dissolved in
NaOH (1 N) then made to volume with H2O.
VI Naphthol (Sigma) 1% in stock alkali, mix new
daily
96