The light reactions result in the production of reducing power, which reduce BADP and in the production of ATP. In the dark reactions, these agents are used to reduce CO2. Until the discovery of radioactive carbon, the intermediates and pathways of synthesis of sugars could only be guessed at. When Calvin and his co-workers supplied 14CO2 to suspensions of alga Chlorella they found that major initial product of photosynthesis was the three-carbon acid, 3 phosphoglyceric acid (PGA). Thus Melvin Calvin (1954) an American scientist worked out the oath of carbon by using paper chromatography technique. In recognition of his work Calvin was awarded with Nobel Prize in 1961. Analysis of plant extracts were made within seconds after beginning of photosynthesis with the injection of 14CO2. For studying the process, illuminated suspensions of Chlorella cells were used. Chlorella was killed at different intervals by dropping the suspensions in hot methanol. Hot methanol also denatured the enzymes. The radioactive substances in alga were separated by two dimensional paper chromatography.
The radioactive spots on chromatogram can be located by radioautography. The chromatogram is placed next to the sheet of X-ray film in a holder for a period of hours or days depending on the intensity of radioactivity. These spots which are radioactive cause a darkening of the film over their location, so that the film records a picture of the position and intensity of radioactivity of the spots on chromatogram.
Various steps of dark phase can be studies as under:
(a) Carboxylation. The first step is the carboxylation of Ribulose 1,5 biphosphate (RuBP) by atmospheric CO2 in the presence of enzyme RuBP carboxylase or RuBisCO. Six molecules of RuBP (5 carbon compound) come in contact with six molecules of CO2 to form six molecules of six carbon unstable compound which is converted into 12 molecules of PGA (3 carbon compound) with the help of an enzyme called carboxydismutase. Rubisco is a large protein molecule and constitutes 16 percent of chloroplast protein and is one of the most abundant proteins on earth.
(b) Glycolytic reversal. PGA is reduced to PGAL (Phosphoglyceraldehyde). This process is the reverse of oxidation step in glycolysis. Total number of PGAL molecules thus formed are 12. The reaction occurs in two steps. Firstly, PGA is phosphorlylated by ATP to 1, 3 diphosphoglyceric acid. Secondly, 1, 3 diphosphoglyceric acid forms phosphoglyceraldehyde (PGAL) with the help of NADPH + H+. From phosphoglyceraldehyde, dihydroxyacetone phosphate (DHAP) is formed with the help of enzyme triose phosphate isomerase. DHAP condenses with PGAL to form fructose 1, 6 diphosphate. From fructose 1, 6 diphosphate, one phosphate group is removed to form fructose 6 phosphate and the step is called dephosphorylation. Finally different types of compounds like glucose, starch are synthesized from fructose 6 Phosphate (F-6-P).
(c) Regeneration of RuBP—F-6-P next undergoes a transketolase reaction that removes the two top carbons as the thiamine pyrophosphate (TPP) derivative of glycoaldehyde, leaving the tetrose erythrose 4-phosphate (E-4-P). The (E-4-P) condenses by aldolase reaction with DHAP to form sedoheptulose diphosphate (SDP), and this is coverted by a second energy-liberating step to sedoheptulose-7-phosphate (S-&-P) and Pi by a phosphatase reaction. As far as can be determined, the same phosphatase works on FDP and SDP. The S-7-P undergoes a transketolase reaction in which the two top carbons are removed as TPP-glycoaldehyde leaving the pentose ribose-5-phosphate (R-5-P). This is converted into ribulose-5-phosphate (Ru-5-P) by phosphopentose isomerase. The TPP-glycoaldehyde derived from F-6-P and F-7-P in the transketolase reaction is transferred to PGA forming xylulose-5-phosphate (Xu-5-P) which is converted to Ru-5-P by a phosphopentose epimerase. The R-5-P is converted to Ru-5-P by an isomerase and is phosphorylated by phosphoribulokinase, ATP being the donor, to produce ribulose biphosphate (RuBP) and ASP (a second “priming” reaction that prepares the pentose for carboxylation).