A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases.
We present here an improvement in the classical molybdate method for inorganic phosphate determination. This method has high sensitivity, with 1 nmol of Pi giving an absorbance change of 0.060 A850 unit. It is highly reproducible and the color remains stable for at least 3.5 h. In addition the use of sodium dodecyl sulfate makes it possible to stop enzymatic reactions without organic phosphate hydrolysis. It also shows an extremely low interference by highly concentrated solutions of different origins. Of special interest is its high tolerance to protein, permitting as much as 50 mg/ml of human serum protein in the sample without precipitation or color interference. For these reasons, it proves to be very useful in the determination of ATPases in tissues such as the ocular lens with low specific activity.
Chifflet
S
S
Departmento de Bioquimica, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay.
Torriglia
A
A
Chiesa
R
R
Tolosa
S
S
D000818:Animals; D001798:Blood Proteins; D003124:Colorimetry; D003459:Crystallins; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology*; D009943:Organophosphorus Compounds; D010710:Phosphates / Q000032:analysis*; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D013055:Spectrophotometry, Infrared
D001798:Blood Proteins; D003459:Crystallins; D009943:Organophosphorus Compounds; D010710:Phosphates; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase