I carried out the superoxide activity SOD as per protocol and took reading at 560nm on spectrophotometer and similarly took absorbance of POD at 470nm. Now how to calculate SOD and POD values from these abosrnace values into IU/mg of protein???? Help me plzz
For SOD activity, you must calculate:
(1º) the maximum percent inhibition of the blank sample / minute: Abs max/min = Absmax (light) - Absmax (dark) / time;
(2º) and of the sample: Abssample/min = Abssample (light) - Abssample (dark) / time;
(3º) Cross-multiply the results from steps 1 and 2: if Absmax/min = 100%, then Abs sample/min = X%
(4º) Percent Inhibition : (I%) = 100 - X%
(5º) Unit of activity (UA): one unit of activity is required to inhibit 50% of photoreduction of NBT to blue formazan. If 50% of photoreduction = 1 UA, then (I%) = Y UA
(6º) To express in mL (UA/mL), correct Y value to 1 mL of sample: for example, if 10 uL was pipetted you must multiply by 100;
(7º) To express in mg of protein (UA/mg P), you must divide the result from step 6 by the protein concentration of the sample.
For POX activity:
(1º) deltaAbs480nm sample = Abs480 sample (final time) - Abs480 sample (initial time);
Ex: deltaAbs480nm sample = Abs480 sample (120 s) - Abs480 sample (20 s) = 100s
(2º) Correct the result from step 1 to minute (Abs480 sample/min);
(3º) Correct the result from step 2 to mL (Abs480 sample/min/mL). For example, if 10 uL was pipetted, multiply Abs480 sample/min by 100;
obs: If the sample was diluted, multiply by dilution factor
(5º) Unit of activity (UA): one unit of activity is required to change one unit of absorbance/mL/min.So,if 1 Abs/mL/min = 1 UA, then Abs480 sample/min/mL = X UA;
(6º) To express in mg of protein (UA/mg P), you must divide the result from step 5 by protein concentration of the sample.
Best regards
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes