Linked enzyme assays

Numerous reactions do not include products or substrates which absorb light at a suitable wavelength.  In this case  it is frequently  possible  to assay  the enzyme  which catalyzes  this reaction  through linking or coupling  it to a second enzyme  reaction which does include a features  absorbance  modification. For instance, the action of the enzyme glucose oxidase,  that is frequently used to measure  the concentration of  glucose  in  the  blood  of  diabetic  patients  doesn’t  result  in  a  change  in absorbance upon conversion of substrates to products which is shown in the figure. Moreover, the hydrogen  peroxide  produced  in  this  reaction  can  be  acted  on  through  a  second peroxidase, enzyme, that concurrently  translates  a colorless  compound  into a colored one (chromogen) whose absorbance can be simply measured.

If the activity of the first enzyme glucose oxidase is to be measured accurately the second enzyme peroxidase and its cosubstrates or coenzymes must be in excess so as not to be the rate-limiting step of the connected assay. This will ensure which the rate of production of the colored chromogen is proportional to