By Oliver H. Lowry
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Additional info for A Flexible System of Enzymatic Analysis
Under 5 μΐ the colorimetric method is required. Calibration of Pipettes 55 Although is it desirable to have available accurately calibrated pipettes, in most analyses this is not essential, since the pipette volumes will cancel out of the final calculation if standard solutions and samples are handled with the same pipettes. The calibration of pipettes is one of the best ways to test pipetting technique. Failure to obtain satisfactory checks means faulty technique or bad pipettes. Since the gravimetric method is the most precise, it offers the most critical test, but only for larger pipettes.
For enzymes with Km9s of 50, 100, 200, and 500 μΜ, the minimal enzyme requirement for [S]0 = 100 μΜ would be 30, 50, 90, and 210 U/liter, respectively, and only 10 U/liter less if [S]0 = 1 μΜ. 2. A Kinetics Primer for the Tissue Analyst 32 When Ku is substantially larger than [S] 0 , Eq. (2-27) reduces to Vm = 4 KJt. Thus, if the KM is 200 μΜ, to complete the reaction (98%) in 20 min would require enzyme activity equal to 40 μΜ/min or 40 units per liter. One-Step Reactions with Two Substrates The majority of analytical reactions to be considered will involve two substrates (A, B).
Colorimetric calibration is also more likely to reveal bad technique since it can be used with the smallest pipettes, for which pipetting faults can cause very serious errors. GRAVIMETRIC CALIBRATION Constriction pipettes are calibrated by delivery of water into a vessel already containing a little water. The increase in weight of the vessel is measured on an analytical balance. A narrow-mouthed vial without stopper and with a capacity not more than 20 times the volume of the pipette is recommended.