PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 181 



Calculation of protein concentration: 



0.87 X 0.273 = 0.237 mg of protein per milliliter in the cuvette 



0.237 X 15 (dilution factor) = 3.585 mg of protein per milliliter of original protein 



solution. 



The sample may be recombined with the stock solution after estimation, 

 since no protein is denatured during the procedure. This feature of the 

 optical method is particularly advantageous at high degrees of enzyme 

 purification when the enzyme solution often contains a very small amount 

 of protein. 



The protein concentration of extracts containing large quantities of 

 nucleic acid (e.g., before protamine treatment) may be estimated by any 

 of several colorimetric procedures. The method of Lowry et al. (1951) 

 utilizing the Folin-Ciocalteau phenol reagent is recommended because 

 of its high sensitivity and is described here. 



Reagents: 



1. 40 g of Na2C03 in 500 ml of distilled water 



2. 0.3 g of CuS04 and 0.6 g of sodium tartrate in 500 ml of distilled water 



3. 1 part of Folin-Ciocalteau phenol reagent (available commercially) and 

 2 parts of distilled water 



Procedure. To a protein sample containing 7-70 jug of protein, add 

 sufficient water to make a total volume of 0.5 ml. Add 5.0 ml of carbon- 

 ate-copper-reagent (equal volumes of reagents 1 and 2 mixed just before 

 use), mix, and incubate at 37°C for 30 min. After cooling to room tem- 

 perature, add 0.5 ml of the diluted phenol reagent, mix, let stand 20 min 

 at room temperature, and read at 660 m/z. For preparation of the 

 standard curve, a satisfactory standard protein solution may be prepared 

 using crystalline bovine albumin (Armour and Co., Chicago). The 

 dilute albumin solution is subject to surface denaturation and should be 

 freshly prepared for each determination. 



BIOCHEMICAL TECHNICS 



Manomeiric Technics 



Manometric methods are employed widely because of accuracy and 

 speed in the quantitative analysis and rate measurements of reactions 

 involving either the evolution or uptake of certain gases. In addition, 

 acid production may be measured by following CO 2 evolution in a bicar- 

 bonate buffer in equilibrium with a CO2 gas mixture. The reader is 

 referred to two excellent books for detailed discussions of the methods 

 and types of apparatus available (Dixon, 1943; Umbreit et al., 194V>;. 

 lliere are two major types of manometers in use, i.e., the constant-pres- 



