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Page Title: Appendix A: Numerical Values in Equation

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Quality Control 4689 Appendix B: Example for calculating IUPAC-FPU

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12. Appendix A: Numerical Values in Equation Used to Calculate Filter Paper Activity

The practical bottom line is that if the assays are set up according to the instructions, and the
calculations are carried o u t using the equation presented in the calculations section, the results
obtained will correspond to the generally accepted activities in " filt er paper units" that wo u ld be
obtained by other laboratories around the wo rld , were these other laboratories to test the same
enzyme solution. For those workers interested in the derivation of this equation, and of the " filt er
paper u n it " , the fo llo win g comments may be h elp fu l in conjunction wit h Ghose (1987).

The numerator (0.37) in the equation is derived fro m the factor fo r converting the 2.0 mg of
"glucose-equivalents" generated in the assay to mmoles of glucose (2.0 ÷ 0.18016), fro m the volume
of the enzyme being tested that is used in the assay (0.5 mL), and fro m the incubation time (60
minutes) required fo r generation of the reducing equivalents.

(2.0 mg glucose / 0.18016 mg glucose/µmol)

= 0.37 µmol/ minute - mL
(0.5 mL enzyme dilution x 60 minutes)

Thus,
Because the "enzyme concentration" in the denominator of the equation is a dimensionless number
(equal to the ratio of the enzyme concentration in the 0.5 mL of enzyme dilution added to the assay
to the enzyme concentration in the original solution, fo r which FPU values are desired), the right side
of equation therefore winds up with units (mmo l min -1 mL-1 ) that lo o k like "International Units per
mL" (I.U./mL). Ghose himself points out, however, that "because the FPU assay is non-linear, the
use of the International Un it per se is incorrect as this u n it is based on in it ial velocities, i.e., linear
reactions in wh ich the product is produced at the same rate during each and every minute of the
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reaction." The author goes on to recommend that FPU values fo r a given cellulase solution be given
simply as "units/mL".

"Definition" of the "Filter Paper Un it " :

As a result of the above choice of numerical values, the "Filter Paper Un it " is not actually explicitly
defined. What is defined is the quantity 0.1875 FPU, wh ich is that quantity of enzyme activity that,
when assayed according to the instructions contained herein, will produce reducing sugar equivalent
to 2.0 mg of glucose. One can v erify this fro m the equation presented in the calculations section by
assuming that the enzyme solution being tested needs no dilution to yield reducing sugar equivalent
to 2.0 mg of glucose (i.e., the "enzyme concentration" ratio in the denominator is equal to 1.0), in
wh ich case the activity of the solution being tested is measured as 0.37 filter paper units per mL.
Inasmuch as 0.5 mL of this solution was used in the assay, the absolute quantity of enzyme activity
that is present in the assay (and to wh ich the observed effect can be ascribed) is 0.1875 FPU.

Procedure #006
Revision: 7/18/96
Supersedes: 8/19/92 Page 8 of 11