Scope
Principle
This assay is based on the enzymatic hydrolysis of the peptide bonds of a defined gelatin substrate at pH 7.0 and 40°. The corresponding reduction in substrate viscosity is determined with a calibrated viscometer. One Viscometric Protease Unit is defined as that activity which will produce a relative fluidity change of 0.01 per sec in a defined gelatine substrate under the conditions of the assay.
Special Apparatus
Calibrated viscometer: Size 100 Calibrated Cannon-Fenske Type Viscometer, or its
equivalent, supplied as Catalog No. P2885-100.
Constant temperature glass water bath (40 ± 0.1°): Constant temperature glass water bath, or
its equivalent, supplied as Catalog No. W3520-10 (Available from Scientific Products, 1210 Waukegan Rd., McGaw Park, Ill., 60085, USA.).
Stopwatches: Stopwatch calibrated in 1/10 min for determining the reaction time (Tr) and
stopwatch calibrated in 1/5 sec for determining the efflux time (Tt).
Reagents and Solutions
Disodium monohydrogen phosphate solution (1 N): Dissolve 47.32 g of anhydrous disodium
phosphate in approximately 800 ml of distilled water in a beaker. Quantitatively transfer to a 1,000-ml volumetric flask and dilute to volume with distilled water.
Monosodium dihydrogen phosphate solution (1 N): Dissolve 40.00 g of anhydrous
monosodium phosphate in approximately 800 ml of distilled water in a beaker. Quantitatively transfer to a 1,000-ml volumetric flask and dilute to volume with distilled water.
Phosphate buffer (pH 7.0): Using a standardized pH-meter, add disodium monohydrogen
phosphate solution (1 N) with continuous agitation to 800 ml of monosodium dihydrogen phosphate solution (1 N) until the buffer is pH 7.0 ± 0.05.
Gelatine substrate (4.0% w/v): With continuous agitation, disperse 20.00 g (moisture-free
basis) of gelatin in approximately 400 ml of distilled water in a 1,000-ml Erlenmeyer flask. The dispersion must be free of lumps. Swell the gelatin for 30 min at room temperature with occasional swirling. Place the gelatin solution on a 40 ± 0.1° waterbath. Swirl occasionally until the gelatin is completely solubilized with no particles appearing in solution. Cool to room temperature and quantitatively transfer to a 500-ml volumetric flask and dilute to volume with distilled water.
Enzyme Preparation: Prepare an enzyme solution so that 1 ml of the final dilution will
produce a relative fluidity change between 0.18 and 0.22 in 5 min under the conditions of the assay. Weigh the enzyme and quantitatively transfer to a glass mortar. Triturate the enzyme with distilled water and quantitatively transfer to an appropriate volumetric flask. Dilute the volume with distilled water and filter the enzyme solution through Whatman No. 1 filter paper, or equivalent, prior to use.
Procedure
Place the calibrated viscometer in the 40 ± 0.1° water bath in an exactly vertical position. Use only a clean viscometer. Cleaning is readily accomplished by drawing a large volume of detergent solution followed by distilled water through the viscometer. This can be accomplished by using an aspirator with a rubber tube connected to the narrow arm of the viscometer.
Pipet 20 ml of gelatin substrate and 3 ml of phosphate buffer into a 50-ml Erlenmeyer flask. Allow at least two flasks for each enzyme sample and one flask for a substrate blank. Stopper the flasks and equilibrate them in the water bath for 15 min. At zero time pipet 1 ml of the enzyme solution into the equilibrated substrate. Start the stopwatch calibrated in 0.1 min and mix solution thoroughly. Immediately pipet 10 ml of the reaction mixture into the wide arm of the viscometer.
After approximately 2 min apply suction with a rubber tube connected to the narrow arm of the viscometer drawing the reaction mixture above the upper mark into the driving fluid head. Measure the efflux time by allowing the reaction mixture to freely flow down past the upper mark. As the meniscus of the reaction mixture falls past the upper mark, start the other stopwatch. At the same time record the reaction time in min from the first stopwatch (Tr). As
the meniscus of the reaction mixture falls past the lower mark, record the time in sec from the second stopwatch (Tt). Immediately redraw the reaction mixture above the upper mark and
into the fluid driving head. As the meniscus of the reaction mixture falls freely past the upper mark, restart the second stopwatch. At the same time, record the reaction time in min from the first stopwatch (Tr). As the meniscus of the reaction mixture falls past the lower mark,
record the time in sec, from the second stopwatch (Tt).
Repeat from redrawing the reaction mixture above the upper mark, until a total of 4 determinations is obtained over a reaction time (Tr) of not more than 15 min.
Prepare a substrate blank by pipetting 1 ml of distilled water into 24 ml of buffered substrate. Pipet 10 ml of the reaction mixture into the wide arm of the viscometer. Determine the time (Ts) in sec required for the meniscus to fall between the two marks. Use an average of 5
determinations for Ts.
Prepare a water blank by pipetting 10 ml of equilibrated distilled water into the wide arm of the viscometer. Determine the time (Tw) in sec required for the meniscus to fall between the
two marks. Use an average of 5 determinations for Tw.
Calculation
One Viscometric Protease Unit (VPU) is that activity which will produce a relative fluidity change of 0.01 per sec in a defined gelatin substrate under the conditions of the assay.
Calculate the relative fluidities (Fr) and the times (Tn) for each of the four (4) efflux times (Tt)
and reaction times (Tr) as follows:
Fr = (Ts - Tw)/(Tt - Tw)
Tn = 1/2 (Tt/60) + Tr = (Tt/120) + Tr
where
Fr is relative fluidity for each reaction time,
Ts is average efflux time for the substrate blank in sec,
Tw is average efflux time for the water blank in sec,
Tt is efflux time of the reaction mixture in sec,
Tr is elapsed time in min from zero time, i.e. the time from addition of the enzyme solution to
the buffered substrate, until the beginning of the measurement of efflux time (Tt),
Tn is reaction time in min (Tr), plus one-half of the efflux time (Tt) converted to min.
Plot the four relative fluidities (Fr) as the ordinate against the four reaction times (Tr) as the
abscissa. A straight line should be obtained. The slope of this line corresponds to the relative fluidity change per min and is proportional to the enzyme concentration. The slope of the best line through a series of experimental points is a better criterion of enzyme activity than is a
single relative fluidity value. From the graph determine the Fr values at 10 and 5 min. They
should have a difference in fluidity of not more than 0.22 nor less than 0.18. Calculate the activity of the enzyme unknown as follows:
VPU/g = [1,000 (Fr10 - Fr5)] / (W x 300 x 0.01) = [333 (Fr10 - Fr5)] / W
where
Fr5 is relative fluidity at five (5) min of reaction time
Fr10 is relative fluidity at ten (10) min of reaction time
300 is time of relative fluidity change in sec from Fr10 to Fr5
1,000 is milligrams per g
W is weight in milligrams of enzyme added to the reaction mixture in a one (1) ml aliquot of enzyme solution
0.01 is change in relative fluidity per sec per VPU.