228 CUVETTE COLORIMETRY 



Lowry and Lopez Method for Inorganic Phosphate in Presence 

 of Lahile Phosphate Esters 



SPECIAL REAGENTS 



Protein Precipitant. 5% trichloroacetic acid, or 3% perchloric 

 acid, or (with very labile esters) saturated ammonium sulfate 

 which is 0.1 A^ with respect to acetic acid and 0.025 A^ with respect 

 to sodium acetate (pH 4). 



0.1 N Sodium Acetate. 



Acetate Buffer (pH 4) . 0.1 X to acetic acid and 0.025 N to sodium 

 acetate. 



1 % Ascorbic Acid. 



1% Ammonium Molybdate in 0.05 N Sulfuric Acid. 



0.05 mM Standard Phosphate Solution. 



PROCEDURE 



1. Deproteinize the sample with ice-cold protein precipitant. If 

 either of the acid precipitants is used, bring the extract rapidly to 

 pH 4.0 to 4.2 by adding 4 vol. of 0.1 .V sodium acetate. (Most labile 

 esters are fairly stable at this pH.) 



2. Dilute the extract with the acetate buffer until the inorganic 

 phosphorus concentration is 0.015 to 0.1 mM (0.05 to 0.3 milligram 

 per cent) . Dilute ammonium sulfate extracts at least five times. 



3. Add 0.1 vol. 1% ascorbic acid and 0.1 vol. 1% acid-molybdate 

 soln. to each vol. of extract. If used within 15 min. of their mixing, 

 the ascorbic acid and molybdate may be combined. 



4. Carry out the colorimetric reading at 5 and again at 10 min. 

 after the addition of molybdate using light between 650 and 950 

 vcijx (maximum absorption at 860 m/x) . 



5. Take simultaneous readings of the standard soln. and blank, 

 which should be prepared parallel with the unknown. Should a 

 difference be observed in the readings of the unknown at 5 and 10 

 min. compared to the standard, extrapolate the values to zero time. 



note: Lowiy and Lopez have found the reaction to be delayed in the 

 presence of certain tissue extracts. In these cases standardization must be 

 obtained by adding a known quantity of inorganic phosphate to an aliquot 

 and using the difference in the readings effected by the added phosphate 

 for the standardization. Dilution overcomes the inhibitory effect to some 

 degree. Thus, brain and muscle extracts should be diluted to a volume 

 150-200 times that of the tissue, and, in the case of liver 300-500 times. 



