The Hill reaction was observed by subjecting chloroplast into three different set-ups, light, dark

and control set-up. An isolation of chloroplast was done by placing 10 g of deveined papaya ( Carica
papaya) leaves on an ice-cold blender with 50 mL of 0.35M cold NaCl solution. The jar was blended at
high speed for 30 seconds then cooled inside the refrigerator for 30 seconds. After three cycles of
blending and cooling, the extract was filtered into an ice-cold beaker through a double-layer
cheesecloth. The suspensions were transferred equally into two 50mL centrifuge tubes. The suspension
were centrifuged at 200 g (1500 rpm) for 5 minutes. The supernate was decanted into another two
50mL centrifuge tubes while the pellets were discarded. The supernate was further centrifuged at 1400
x g (4000 rpm) for 15 minutes. The supernatant was discarded while the pellets were resuspended with
5 mL 0.35 M NaCl to each tube. The suspensions were placed on a small beaker immersed in an ice bath.
After isolation, the extraction of chlorophyll was performed by diluting 0.1 mL of the prepared
chloroplast suspension was to 19.9 mL of 80% acetone. A blank cuvette containing 5mL of 80% acetone
was first read in a spectrophotometer set at a wavelength of 652 nm before the 5mL chlorophyll
solution was read. Thee dilution was discarded after the absorbance reading. The amount of total
chlorophyll per mL of the chloroplast suspension was calculated. Moreover, the original suspension was
diluted with the proper amount of cold 0.35 M NaCl solution to obtain a concentration of 0.05 mg
chlorophyll/mL.

As for the spectrophotometric measurements of the Hill reaction, 15 mL of the 0.05 mg

chlorophyll/mL were placed in a test tube wrapped with aluminum foil and chilled in an ice bucket as the
source of unheated chloroplast. The remaining suspension was placed on another test tube and then
heated with an alcohol lamp until boiling. The heated mixture was allowed to stand for 5 minutes for the
settling of debris. Six test tubes were prepared in which two served as blanks (Control and Experimental)
while the remaining were used for the experimental set-ups (Control, Light and Dark). As for the blanks,
1.5 mL of 0.2 M phosphate buffer and 2mL of 0.35 M Nacl were added. The only difference in the control
and experimental blanks was the addition of 1mL of heated chloroplasts and unheated chloroplasts,
respectively. In the experimental set-up, all three tubes were poured with 1.5mL of 0.2M phosphate
buffer and 1mL of 0.35 M NaCl. The control set-up has additional 1 mL heated chloroplasts; the light set-
up with 1mL of unheated chloroplast; and the dark setup with 1mL unheated chloroplast with the tube
covered with aluminum foil. After that, all tubes were immediately added with 1mL of DPIP before the
first reading. The spectrophotometer was set at 605 nm wavelength and the appropriate blanks were
zeroed on the machine. The initial reading of the control and experimental set-ups were recorded. As
for the dark tube, the cuvette was immediately wrapped with aluminum foil after the first reading and
allowed to stand for 30 minutes for the last reading. On the other hand, the control and light tubes were
placed in an ice bath with 100-watt bulb illumination place 18 inches away after every reading. These
tubes were read at a 5-minute interval for 30 minutes.