1.

Protein estimation

The interaction between bio molecules is crucial for knowing their function. But the presence
of the protein impurities can highly affect the result. Therefore, it is important to know the
total protein concentration that includes both active and inactive forms of protein. Lowry
method (1951) is one of the important methods of protein assay for determining the total
amount of protein present in a given sample. So, the basic objective of this test is to estimate
the amount of protein present in a sample by Lowry’s method.

Principle: The principle involved in Lowry method is determining the protein concentration
by calculating the oxidation of the peptide nitrogen with the Copper ions under alkaline
conditions followed by reduction reaction of Folinciocalteay phosphomolybdic
phophotungstic acid to Heteropolymolybdenum blue by copper catalyzed oxidation of
aromatic acids. However, the method is highly sensitive to pH of the assay solution for which
it should be strictly maintained at 10-10.5. But if very small volume of sample is used then
pH will have least effect. Some other substance that affects Lowry procedure is buffers,
sugars, nucleic acids, zwitter, lipids and Sulphydryl reagents. Therefore, care should be taken
to see that these substances are removed before starting the experiment.1

Reagent required
Reagent A -2% Na2CO3 in 0.1 N NaOH.
Reagent B -0.5% CuSO4.5H2O in 1% sodium or potassium tartarate.
Reagent C - 50 mL of Reagent A was added with 1 ml of Reagent B
Reagent D -Folin Ciocalteau solution + distilled water (1:2 ratio).
500 mg of the sample was weighed and grounded well with a pestle and mortar in 5 ml
of the buffer (0.2 M phosphate buffer of pH 7) solution. This solution was centrifuged and the
supernatant was used for protein estimation. 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 ml of
the bovine serum albumin (BSA) for working standards were pipetted into a series of test
tubes. 1.0 ml of the sample extract was pipetted into other test tube. All the test tubes were
made up to 10 ml with distilled water. 10 ml of alkaline copper sulphate reagent (reagent C)
was added to it. The solution was mixed well and incubated at room temperature for 10 min.
After incubation, 0.4 ml of reagent Folin Ciocalteau solution (reagent D) was added to each
tube and incubated again for 30 min which developed blue colour of solution. The optical
density of solution was recorded at 660 nm. Content of protein was measured against the
standard curve prepare from BSA and expressed as mg protein g−1 fw of leaf.
2. Ascorbic acid
Ascorbic acid otherwise known as Vitamic C is antiscorbutic. It is present in citrus fruits,
gooseberry, bittergourd etc. in high amount. Generally it is present in all fresh vegetables and
fruits. It is water soluble and heat-labile vitamin. The method described below is easy, rapid
and a large number of samples can be analyzed in a short time.

Principle

The dichlorophenolindophenol (DCPIP) (a blue solution in oxidised state) solution is reduced

by ascorbic acid to a colourless solution (DCPIPH2). During this process, the ascorbic acid
in-turn is oxidized to dehydro-ascorbic acid, which in acidic medium, gives a light pink
colour.

Reagent required

Extracting solution- 0.4% of oxalic acid

DCPIP-20 µg ml-1
Purified ascorbic acid
Ascorbic acid was estimated using 2,6-dichlorophenol indophenol (DCPIP) reduction
method of Keller and Schwager (1977). Briefly, 0.5 g fresh weight of leaf was homogenized
with 20 mL of ice cold extracting solution. After centrifugation at 6000x g for 15 minute, 1
ml of sample supernatant was mixed with 5 ml of DCPIP at constant shaking. The absorbance
of developed pink colour was recorded at 520 nm. Developed pink colour was bleached by
adding one drop of 1% ascorbic acid and the absorbance was recorded at the same
wavelength. The observed value was compared with a standard curve prepared using known
concentration of ascorbic acid and the result was expressed as mg As g-1.

500 x V 2 x 25 x 100
Amount of ascorbic content (mg/100 g)=
V 1 x5 x5

Where; 500 = µg of standard ascorbic acid taken for titration, V1 = Volume of dye
consumed by 500µg of standard ascorbic acid, V2 = Volume of dye consumed by 5 mL of
test sample, 25= Corresponds to total volume of the extract, 100 = Ascorbic acid
content/100g of the sample, 5 = Weight of sample taken for extraction, 5 = Volume of the test
sample taken for titration