The Biosynthesis of Metal Porphyrins by Subchloroplastic Fractions

Cucumber seeds (Cucumis sativus L. cv. Alpha Green) were germinated in vermiculite (Terra Lite) at 24 C in complete darkness for 4 days (7, 8). Cotyledons were harvested with full hypocotyl hook as previously described (2). They were placed in beakers with enough distilled H20 to keep them moist and were irradiated with white fluorescent light for 4 hr (8). Developing chloroplasts were isolated from the greening cotyledonary tissues. Five grams of cotyledons were ground in 7.5 ml of fortified 0.2 M tris-HCl, 0.5 M sucrose, pH 8.0, containing 37.5 ,umoles of GSH, 2.25 ,umoles of CoA, 3.75 ,umoles of MgCl2, 375 1tmoles of potassium phosphate, 180 ,umoles of methanol, 3 ,umoles of ATP, and 0.45 ,umole of NAD. The slurry was filtered through four layers of cheesecloth, and the resulting crude homogenate was centrifuged at 0 C for 3 min at 200g. The supernatant was centrifuged for 7 min at 1500g, and the resulting chloroplast pellet was lysed in 21 ml of fortified 25 mm tris-HCl, 25 mm potassium phosphate buffer, pH 7.7, lacking sucrose but containing the same concentration of GSH, CoA, MgCl2, methanol, ATP, and NAD as the homogenization buffer. The suspension was kept on ice in the dark for 15 min then centrifuged at 105,000g for 60 min in a Spinco No. 40 rotor. This centrifugation separated the lysed plastids into a water-clear soluble protein fraction (S3) and a compact, green, particulate fraction (P3). S3 was withdrawn with an eye dropper. and P3 was suspended in 21 ml of the fortified lysing buffer. Two milliliters of S3, P3 and S3 + P3 (1 :1 v/v) were incubated in glass tubes on a metabolic shaker with 25 pImoles of ALA.3 The incubation was carried out at 28 C under 5 ft-c of white fluorescent light with moderate shaking speed for 16 hr. Three milliliters of the reaction mixture at pH 7.7 contained: 150 ,tmoles of tris-HCl, 100 ,tmoles of sucrose, 100 1amoles of potassium phosphate buffer, 1 /Imole of MgCl., 10 Imoles of GSH, 0.6 ,tmole of CoA, 1.2 mmoles of methyl alcohol, 1 ,mole of ATP, and 0.15 ,tmole of NAD. The reaction was stopped by the addition of 15.0 ml of acetone:O.1 N NH4OH (9:1 v/v) and centrifuged at 39,000g for 10 min. The pellet was saved for the extraction of free porphyrins and the acetone: NH,OH supernatant containing metal porphyrins and free porphyrins was extracted with an equal volume of hexane. The hexane extracted most of the /-carotene and chlorophyll. The metal porphyrins remaining in the hexane-extracted acetone: NH4OH supernatant were transferred to ether as previously described (6). The proteinaceous pellet and the aqueous acetone phase that was left after metal porphyrins extraction were pooled and used for the extraction of free porphyrins as described previously (6). Free porphyrins were separated by ascending paper chromatography on Whatman No. 3 MM paper in 2,6-lutidine:0.05 N NH4OH (5:3.5 v/v). Metal porphyrins, in ether, were separated on thin layers of Silica Gel H in benzene-ethyl acetateethanol (8:2:2 v/v). All fractions were routinely monitored by their absorption spectra and recorded with a Beckman double beam spectrophotometer Model DK-2A. All absorbancies used in quantitative determinations were derived from the appropriate spectra. The amount of uroporphyrin in acidified, saturated sodium acetate (pH 2.5) was estimated from absorbancy at 402 nm using the molar extinction coefficient (5.41 X 105) and the correction factors reported by Rimington and Sveinsson (10) for uroporphyrin in 0.5 N HCl. The copro + protoporphyrin fraction in ether containing mostly coproporphyrin was estimated from its absorbancy at 401 nm and a molar extinction coefficient of 1.8 X 10' (6). When protoporphyrin was predominant in this fraction, its absorbancy at 404 nm and a molar extinction coefficient of 1.5 X 10' were used instead (6). The amount of metal porphyrins in ether and in the presence of some chlorophyll and protochlorophyll contamination was estimated as "Mg protoporphyrin equivalent" by the following equation: nmoles per ml = 56.61 A,. 13.24 A6247.79 A,48 where A..S, A,2,, and A64, represent the absorbancies of the ether extract at 583, 624, and 648 nm respectively. The above equation was derived from the molar extinction coefficients of Mg protoporphyrin, protochlorophyll and chlorophyll a + b in ether at 583, 624, and 648 nm (Table I).