[eccles1214]

How can I convert enzyme activity per minute into micromoles per minute of product produced given that the enzyme was diluted in a 1:70 ratio?  I have done it two different ways to do it, with different results (by a factor of 10).

Here is what I have:
An enzyme was diluted in a ratio of 1 part enzyme to 69 parts buffer.

A  1 cm wide, 1 mL volume cuvette containing 10 microliters of an the 1:70 enzyme solution was placed into a spectrophotomer and the absorbance read was 0.6630/minute.
The molar extinction coefficient of the enzyme is 14250 M^-1 cm^-1 (i.e. L/M cm^-1).  The pathlength is 1 cm.  The cuvette holds 1 mL.  And I converted the L/M to milliliter and millimoles.

My calculations are using Beer's Law A=ecl:
  (:delta:Absorbance/extinction_coeff M^-1 cm^-1)*(mmole/(milliters * cm))*(1 cm pathlength)*1 mL)*(1000 micromole/mmole) = 0.046526.

But what about the 1:70 dilution factor and the fact that the cuvette holds 1 mL but only 10 microliters was put into it so it is further diluted by (1 mL = 1000 microliters; 1000 microliters/10 microliter sample ==> 100)?

So the other calculation would be nearly the same except:
(:delta:Absorbance/extinction_coeff)*(mmole/(ml cm))*(1 cm pathlength)*(1 mL)*(1000 micromole/mmole)*(10 microliters/1000 microliters) = 0.00046526.

The enzyme has a density of 1 mg / 1 mL, so the 1:70 dilution is (1/70)*(1 mg/mL) = 0.0143.  Where does this come into the figuring?

The ultimate goal is to get the absorbance/min into micromoles/min.

[eccles1214]

Do I need to know the MW of the enzyme on a calibration curve to solve this?