206 



A. L. GRAFFLIN, S. OCHOA 



VOL. 4 (1950) 



OPTICAL TESTS AND ENZYME UNITS 



Over-all Reaction. — The activity determination is based on Reaction i. The early rate of 

 reduction of triphosphopyridine nucleotide (TPN) in the presence of enzyme, Mn+"'", and an excess of 

 isocitric acid, is proportional to the concentration of the enzyme within certain limits. The measure- 

 ment is carried out in the Beckman spectrophotometer at wave-length 340 m^ using either glass or 

 silica cells of i.o cm light path. One enzyme unit was defined as the amount of enzyme causing an 

 increase in optical densit}' of 0.0 1 per minute calculated for the third 15 second period after the start 

 of the reaction. 



The reaction mixture, in a final volume of 3.0 ml contained 0.025 ^ glycyl-glycine buffer pH 

 7.4, 0.6- 10-^ M MnClg, 0.45 • 10-* M TPNqx, enzyme, and 0.175 • 1°"^ M rf,/-isocitrate. The volume was 

 made up with water adjusted to a temperature of 22-23°. The blank cell, for setting at 100% light 

 transmission, contained all the above components except TPN. The reaction was started, after taking 

 a zero time reading, by addition of either enzyme or isocitrate. The presence of phosphate in concen- 

 trations higher than 0.0003 M should be avoided because turbidity, due to precipitation of manganous 

 phosphate, may develop. Typical results obtained with an extract of washed acetone-dried pig 

 heart containing 6.0 mg of protein per ml are shown in Table I. 



TABLE I 



OPTICAL TEST FOR REACTION I 

 PROPORTIONALITY OF RATE TO ENZYME CONCENTRATION 



The protein content of the enzyme solutions was determined spectrophotometrically by mea- 

 suring the absorption of light at wave-lengths 280 and 260 m//. The protein concentration was cal- 

 culated from the absorption at 280 m/^ with a correction for the nucleic acid content from the data 

 given by Warburg and Christian®. 



Oxalosuccinic Carboxylase. — The oxalosuccinic carboxylase activity (Reaction 3) was deter- 

 mined by means of a rapid and sensitive optical test. The test is based on the fact that, in the presence 

 of Mn"*""^ and oxalosuccinate, the enzyme causes a pronounced increase in the absorption of light at 

 the wave-length 240 m/i, presumably as a result of increased formation of an intermediate oxalo- 

 succinate-manganese complex ; this increase is followed by a rapid drop indicating decarboxylation^. 

 The early rate of increase of light absorption is, within certain limits, proportional to the concentra- 

 tion of enzj^me. The measurement is carried out in the Beckman spectrophotometer using silica cells 

 of 1.0 cm light path. One enzyme unit was defined as the amount of enzyme causing an increase 

 in optical density of o.oi per minute calculated for the first 15 second period after the start of the 

 reaction. 



The reaction mixture, in a final volume of 3.0 ml, contained 0.134 ^^ potassium chloride, enzyme, 

 0.167-10-^ M MnClg, and approximately 0.167-10-^ M oxalosuccinate*. The volume was made up 

 with water adjusted to a temperature of 15°. The blank cell contained no oxalosuccinate. The reaction 

 was started by addition of oxalosuccinate, which was blown into the mixture from a Lang-Levy 

 micropipette^, and readings of the optical density were made at 15 second intervals thereafter for i 

 or 2 minutes. The optical density of the oxalosuccinate was determined separately and furnished the 

 zero time value. The amount of enzyme was so adjusted that an increase in optical density not below 

 0.07 nor above 0.20 was obtained in the first 15 seconds. The reason for the presence of potassium 

 chloride is that it was found to increase the activity of the enzyme. This effect appears to be a non- 

 specific one caused by the increased ionic strength'. The presence of phosphate in concentrations higher 

 than 0.0003 M should be avoided for the reasons already stated. Typical results obtained with the 

 acetone powder extract of pig heart are shown in Fig. i. , 



* Prepared as previously described*. 



References p. 210. 



