Experimental Design:
1.
Humidity Levels
The goal
in establishing this experiment was to explore possibilities for the alteration
of rice to better protect stores in developing countries where spoilage
contributes significantly to malnutrition. Therefore, it was determined that the
conditions to which the rice used were exposed should be similar to those
conditions existing in such countries. Upon research, it was seen that in many
Asian countries where rice provides the majority of sustenance for the people of
those countries, that humidity rates were very high. In order to mimic these
high rates of humidity, a method for altering the humidity of the rice grains
was developed. A time control was also established, in which the protease
activity after both one and seven days exposure to the humidifier was measured.
Materials:
- saturated salt solution; potassium chloride (83 %) or sodium chloride
(75%)
- 0.5 ml centrifuge tubes
- serum vial (including rubber septum and metal top)
- incubator set at 37 º C
- rice samples
Process:
i.
top of a centrifuge tube was cut off
ii.
tube was filled with rice and the mass of rice in the
tube was determined
iii.
2 ml of the solution (water, KCl, or NaCl) was pipetted
into a serum vial and the centrifuge tube with rice was placed inside a vial
iv.
vial was sealed using a rubber septum and metal top
v.
vial was placed in incubator for either one day or
seven days
The initial use of the assay
showed little or no protease activity in the dry samples. To solve this problem,
the existing method was altered to allow for more enzyme activity. One step that
was significantly altered was the preparation of the samples to allow for more
concentrated amounts of enzyme.
Materials:
- rice samples
- mortar and pestle
-
buffer (100 mM ammonium
bicarbonate)
-
15 ml nylon tubes
-
1.5 ml centrifuge tubes
-
vortex
-
centrifuge
Process:
i.
rice with different rates of humidity was removed from
the incubator and the new mass of each was determined
ii.
rice samples were ground using a mortar and pestle
until they formed powder
iii.
rice powder was placed in a 15 ml nylon tube, and
buffer was added using the ratio one gram of rice powder to five ml of buffer
iv.
rice and buffer were mixed using a vortex
v.
aqueous layer was removed and pipetted into 1.5 ml
centrifuge tubes
vi.
tubes were placed in the centrifuge at 13500 rpm for 5
minutes
vii.
aqueous layer was used as ‘sample’
for the assay
3.
Azocasein Hydrolysis Assay
After the initial use of the
azocasein assay, it was determined that the amounts of protease were not
sufficient to garner clear readings. Therefore, the assay was altered to allow
for five times the amount of enzyme.
Reagents:
-
buffer (100 mM ammonium bicarbonate)
-
substrate (25 mg azocasein in 5 ml of ammonium
bicarbonate buffer)
-
trichloroacetic acid ( 0.2 M ), TCA
-
sodium hydroxide ( 0.5 M ), NaOH
Process:
i.
50 µl of sample was pipetted into labeled tubes (50 µl
of buffer for controls)
ii.
125 µl of substrate was added to each tube
iii.
samples were incubated at 30ºC overnight
iv.
50 µl of TCA was added to each sample to stop reaction
and each was placed on ice
v.
samples were centrifuged at 13 500 rpm for 5 minutes
vi.
100 µl of supernatant was transferred to 96-well plate
vii.
100 µl NaOH was added to each of the wells
viii.
plates were read at 450 nm
4.
Protein Determination
In order to ensure that the
amount of protein in each cell of the gel was equal, protein determination was
performed on each of the different types of rice. The assay used is known as the
Bradford assay, an indicator that makes it possible, upon reading the samples in
the mass spectrometer, to find the level of protein in each sample. Once the
levels of proteins were established, we knew the correct amount of sample to use
in our gels.
- dilute 1 part Bradford
Reagent with 4 parts distilled water
- filter Bradford reagent and
store in opaque plastic bottle at room temperature
- prepare dilutions of
sample, water and bovine globulin
- pipet 5μl of each
dilution in each microtiter plate (done in triplicate)
- add 250 μl of Bradford
reagent into each well
- read in spectrometer at 50
nm
5.
Zymography
Through
zymography, the charge of each protein is equalized so that the only difference
becomes the size of the molecule; larger molecules remain closer to the top of
the gel while smaller proteins remain nearer to the bottom of the gel.
Comparison samples (proteins) are of a known size and can therefore be
used to estimate the size and type of protein in the rice.
Separating Gel
Reagents:
- trisaminoethane hydrochloride ( 1.5 M, pH 8.8 ), Tris HCl
- sodium dodecyl sulfate ( 0.1
M), SDS
- acrylamide/bis (0.3 M )
- ammonium persulfate ( 0.1 M), APS
Process:
i.
2.35 ml ddH2O, 2.5 ml Tris HCl, 100 µl SDS,
4.0 ml acrymalide/bis were mixed
ii.
degassed for 15 minutes
iii.
1.0 ml gelatin (50 mg gelatin in 5 ml ddH2O),
50 µl APS, 5 µl TEMED were added
iv.
glass plates were filled up to ~1 inch from top and
then add 0.5 ml of ddH20 to flatten gel and avoid air contact
v.
polymerized for 35-40 minutes
Stacking Gel
Reagents:
- trisaminoethane hydrochloride ( 0.5 M, pH 6.8 ), Tris HCl
- sodium dodecyl sulfate ( 0.1 M), SDS
- acrylamide/bis (0.3 M )
- ammonium persulfate ( 0.1 M), APS
Process:
ii.
50 µl APS and 10 µl TEMED was added
iii.
remainder of space was filled with stacking gel
solution and comb was inserted
iv.
polymerized for 35-40 minutes
v.
samples were loaded in bin of ice using the MW markers
(marker ingredients: phosphorylase B, bovine serum albumin, ovalburim, carbonic
anhydrase, soybean tripsin inhibitor, lysosyme)
vi.
gels ran at 150 V until the dye front was around 1/8
inches from the bottom of the plate
vii.
gel washed in wash buffer 3 times for 10 minutes each
(see appendices)
viii.
gel incubated in incubation buffer at 30 ° C for 19
hours (see appendices)
ix.
gel stained in coomassie blue stain for 30-35 minutes
on shaker (see appendices)
x.
coomassie blue poured off gel and replaced with destain
for around 40 minutes until desired contrast was obtained (see appendices)
xi.
destain was removed and gels were scanned
6. Inhibitors
The purpose of doing this research was to attempt to find some form of
inhibitor to reduce the amount of enzyme activity in the rice. Several known
inhibitors were chosen to be used as references for the one food grade inhibitor
being used: citric acid. The same azocasein assay as seen above was used, with
only one additional step.
Reagents:
- 5 ml 1% citric acid solution
- 0.5 ml 5 mg/ml aprotinin
- 1.0 ml 1.0 mg/ml pepstatin
- 4.0 ml 500 mM iodoacetamide
- 3.0 ml 500 mM
EDTA
Process:
i.
the stated amount of each protease inhibitor was added to the substrate
and sample and incubated overnight