The Biosynthesis of Metal Porphyrins by Subchloroplastic Fractions

Abstract

The biosynthesis of Mg protoporphyrin monoester, protochlorophyll and chlorophyll a and b by crude homogenates, isolated etioplasts, and differentiating chloroplasts has recently been described by . In the present communication we wish to report briefly on the capacity of subchloroplastic fractions to form free porphyrins and metal porphyrins. MATERIALS AND METHODSCucumber 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). De The reaction was stopped by the addition of 15.0 ml of acetone:0.1 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: NH4OH supernatant containing metal porphyrins and free porphyrins was extracted with an equal volume of hexane. The hexane extracted most of the ,8-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 106) and the correction factors reported by Rimington and Sveinsson (10) for uroporphyrin in 0.5 N HCI.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 105 (6). When protoporphyrin was predominant in this fraction, its absorbancy at 404 nm and a molar extinction coefficient of 1.5 X 105 were used instead (6).

Protochlorophyll and Chlorophyll Biosynthesis in Cell-Free Systems from Higher Plants

“…Carell & Kahn (18) showed that a substantial propor tion of the chloroplastic enzyme remained bound to the membranes of lysed and washed mature Euglena chloroplasts. On the other hand, Rebeiz et al reported that most of the free porphyrin biosynthetic activity of greening etioplasts was found in the soluble fraction (supernatant of the centrifugation at 105,000 g) of the lysed plastids (127). The green membrane fraction exhibited very little por phyrin biosynthetic activity when incubated with ALA.…”

Induction of Porphyrin Synthesis in Etiolated Bean Leaves by Chelators of Iron

“…was vacuum-filtered through an asbestos-coated funnel, and the residue was resuspended volume of the extraction solution and filtrates were combined and the residue, fluorescent, was discarded. The combined with an equal volume of petroleum ether [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] to remove Crystalline standards of copro and proto methyl esters were hydrolyzed in 3 N HCl for 12 hr. This yielded a mixture of partial esters as chromatographic standards (20).…”

Biosynthesis and Accumulation of Microgram Quantities of Chlorophyll by Developing Chloroplasts in Vitro

“…4 (3,17,18). The same appears to be true whenever the normal flow of the chlorophyll biosynthetic pathway is blocked or damaged by incubation in the absence of cofactors (Table III) or by lysis and fractionation of the developing chloroplasts (16). So far it has been possible to show a net chlorophyll biosynthesis in vitro for limited periods of incubation.…”

“…Chlorophyll a + b was prepared from greening cucumber cotyledons and purified by thin layer chromatography as previously described (10). A subchloroplastic soluble enzyme system (S3) capable of uro, copro, and protoporphyrin biosynthesis and accumulation was prepared from developing chloroplasts (16). It was incubated with 25 ,imoles of ALA and cofactors for 16 hr, as described in a previous communication (16).…”