Carbon Dioxide & the Hill Reaction

Abstract

Recently a i uniber of investigators have reported that the Hill reaction is mior-e ral)i(l in the plreelice of CO., thiani in its absence. However., thle phenome-1on1 (it CO., dependence can only Ie (lemonstrated un-(ler certain conl(litioiis and it is thlerefore regrettable that there hlas beenino agreement as to the nlattire of these cond(litions. \Warburg an(l Kiippahl (9) haxve stresse(l the iniportaince of preillumiinatinig the chlioro-pllasts. whereas Abeles et al. (1) foulid the effect primarily in unbufferedl nedia. Stern anid Vennes-land, on the othier han(l, observe(l large CO., effects in butfered niie(lia cand(without preillumination: in thieir stu(dies the composition of the buffer xvas imii-portant (7) \Ve have con fir-mie(d and extendledl the obls ervations of the last-named authors. \\e have ailso invxestigate(l the relati,on of CO., elpendlence to lighit intensity, the state of uncouplilig of the plios-iPhorvlation system, ain(l the niattul-e of the Hill re-atctionl oxidant. The new information l)resenitedl her-e provides little support for \Warburg's contenitioni that CO, pllaYs a role in the Hill reaction atkin to its role ini pihotosynthesis. In fact it is inconsistent with somile of the (letails of Warburg's p)ostulate(l miechiai-isin. Ho ever, the crucial (Iuestioln reniais Un-iswere(l: Is CO.) a metabolite in tile Hill reaction (Or are We (lealiig with ind(lirect and possibly ulispecific effects of the bicar-b-onate ion ? Materials & Methods (Illoroplasts vere isolated frolni voting pea plants (Pi'sum1 stivizum L. var. Alaska) 1b the following proce(ldure: Thle leaves xxwere g round ill a cliilledi iortar with a buffer containing 135 g sucIose, 2.5 g' bovine serum adbumiin, and 0.05 milole tris (hN'droxv-iethl\l)miiethi-lglNcine-NatOH per liter,)H 7.5. The lioniogenate was filtered thlroughi glass wvool inito ice-encaseld centi-ifuge tubes. It was tileni centrifuged ait 4000 X q for 3 miinutes and the supernatant flui(d distcardedl. The chloroplasts xvere resus.pended in thle s;ame buffer., recenltr-ifuge(l, takemi tl) ill (a Smllall vol-tuinme of the buffer all(n store(d in anl ice bath. Simall .aliqIuots of tthe (lense suslemisiosi were ad(l(le(l to tlle reaction Vessels. Oxygen lpro(luction was nmeare(l au ediaiometricallyv in tand(lar(l Warl-urg vessels. P'otassiunm ferricva-Receivedl Aug. 31, 1962. 2 Present address: Department of Botainy anid Planit Pathology, Michigan State lUnliversity, East Lansing, M ichi-aii. iiide, if used, was a(l(le(l fromil the side a.rimi toxwards the end(of the 2 to 3 hour incubation pelrio(l. The preilncubatioll took lplace in the dim light of a shut-tere(l roolim. \hlite lighit frlomii six 300-xv incan(les-cenit r-eflector lamps xx as use(l (tlu-ing the reactionl. The intensity of the lighit, xvhich was iiot measured, fell appreciably sliort of saturaiting thle miiol-e active Hill reactions (see fig 5). The CO.) colicentrationi of the gas phase vas regulate(d 1y the addition of CO., buffers to the center well. Tlle CO., pressures in equilibrium wxith several of thle buffersx%vere mneas-ure(l \xvitlh a miiass spectrometer. These observed pressures agree(l reasonably x-ell xv ith the plressures rel)orte(l by \Warburg anid Krippail (8) and \x-itlm the l)pressures p)re(licte(l by the lax of mass action. Because ferricvanide redluction in the FHill reac-tiOli leads to hvdrogeI ionl formation, a(le(qtIate CO., buffering and pH bufferinig are essential if the re-actioli is to be folloved nianonietricallv. Ini order to iimake the CO., buffer capacity as large as possib)le. the CO., buffers wxere very concentrated (usually about 2.0 m) a(ind 0.4 niil instea(l of the usual 0.2 nil xvas use(l. Spilling oxer of this larger x'olunie x-vas plrexenite(l 1v the use of extra large paper fans ill the center wells. Equilibration of tile CO., huffer with the gas philse xvas catalyzed by the inlusioni of 0.1 _m so(liuiii arsemiite. [Arsenite catalyzes the lh-dlratioll of CO., (6(). In sonile experiments CO, piressue xwa5s miaintaine(l by-a 2.0 xi KHCO 2.0 M so(diumii arsenite soltution milixedl with 1.0(m (diethaiaiol-aminie. This grave 0.6 c, CO., in the ga lphiase ani(l xveriy rmapi(l equilibration. \Wlien sucll plrecautions xxere takeni. it xv-as possible to assXigil practically all of tihe pressure changes in the vessels to 0... Simice there Nxvere somiie aniomlalies ili the reactionl of the chiloroplasts to thle available 2,6-dichiloroplhelnol-in(lopheiiol (DCPIP) purcliase(l frolii the Eastmiiali Organic Chemicals, it xvas suspectedl that the (hIve miglit contain imlpiurities. Conse(quenitly it xas purl-fie(d in the folloxviigi manner: by caieftul regmulation of tile pH! the (lye xvas transferred repeatedly be-tweeni chilol-oforiii (aci(d fornil) ami(l x ater (salt form-). theni betxveen ether and xxater. TIle last traces of ether-xere remoxve(l fromi the filial xvater plhllse by aeration ani(l the sodliumli salt of thle (lye xvas precipi-tate(d by tile a(l(itioni of colitrolle(d aniouiits of NaC1. This saltinig out l)roce(lure xvas repeated. The pUri-fie(d material and the original sampimile had the samile optical (denisity aiid tile sanime effects oil the Hill re-acti mll. Tris(lh\d(r-oxniiietli\l)niiethvlgl-ciine (tricinie) xwas 298 GOOD-CARBON-DIOXIDE & HIILL REACTION2 prepared by reacting tris (hvdroxymethyl) amiiiomoieth-anie (tris) with nmonochloroacetic aci(d as dlescribe(d elsewhere (3). Results Initertactionis of Bicarbonate with Oticr-A nions. The Hill reaction is influencedI very ilmuch by the presence or absenice of a number of different aniions. \\arburg first demlonstrate(l that chloride could be essential to the photochenmical activities of isolated chloroplasts (11). Subse(ouentlv, it was showl that clhlori(le interacts with other anionis in (liverse ways, somletimes stimiulating an(d sometimes inhibiting the Hill reaction, anid that anionis other thaln chloridle re-act in a sinmilar complex manner (3, 5). Consequent-ly it seemed that the dependlence of chloroplasts on CO., (which is miiostly in the fornm of bicarbonate ion at the pH's used) miight be very much miiodified by other anions. Perhaps some other aniioIn could fuil-fill the function of bicarbonate in CO. (leplete(l cliloroplasts, thereby restoring the Hill reactioin po-tential. The results of a large and heterogeneous assort-menit of experimiients, in which chloroplasts were pre-incubated for two to three hours witlh a variety of anions or niiixtures of anions, nmay)e summarize(l as follows: The smaller mionofunctional ionls such as formiate, acetate, chloride. and fluoride, have the effect of in-creasing the dlepen(lence of the chloroplasts on CO.. The saiiie ions are only slightl) inhibitory, if at all.