[stads_29]

If the enzyme creatine kinase is assayed:
                      CK
creatine + ATP -----> Creatine-p +ADP
                     <----

               PK
ADP +PEP ---->ATP + pyruvate
              <----
                        LDH
Pyruvate +NADH ----->lactate +NAD^+
                       <-----

oxidation of NADH is monitered at 340nm (where NAD+ doesnt absorb) In a cell of path length 1cm the absorbance of a 1mM sol od NADH at 340nm is 6.22

If .02ml of a sol of creatine kinase (conc:.08mg/ml) is added at 25C to a 1ml of assay mixture, in a cell of path length 1cm. Decrease in absorbance at 340nm is .52 per min. Whats the activity of the Creatine kinase sol in umol substrate consumed per min per mg enzyme?

[renge ishyo]

As a hint to get you started, consider that you are only measuring the absorbance for the third (LDH) equation. You have a starting concentration of NADH that is given. This is the only species being monitored by the spectrometer. When you add the CK, the reaction starts to go forward and NADH is used up to form NAD+. You monitor NADH being used up as a decrease in absorbance over time (.52 min^-1). What they want you to do is convert this absorbance measurement into a decrease in the concentration of NADH per minute as opposed to a decrease in absorbance per minute. Then once that is done, simply divide this measurement by the amount of CK you added to the mixture in mg (Gah! That's the whole problem!).

[stads_29]

So is the answer 8172.5 with340/.52 = 653.846/.08 = 8172.5?

[renge ishyo]

340 nm is the wavelength of the spectrometer, not the concentration of NADH that you started with. First you have to use A = elc and the given data to find e. Then use can use this e to convert the absobance of .52/time into concentration NADH/time using this same A=elc formula. Then you need to divide this by the mg of enzyme. 0.08 is NOT the mg of the enzyme it is the concentration. You have to multiply 0.08 by the volume of the enzyme you added to the mixture to get the units to be mg.

Use dimensional analysis and do each step carefully one at a time. If your units don't come out then you've done something wrong.

[stads_29]

is the answer then 31100mg?