Hydrolysis Of Chlorophyll Research Articles

Chlorophyllase‐Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

AbstractChlorophyllase converts chlorophyll and pheophytin into their colorless derivatives (chlorophyllide/pheophorbide and phytol). This activity can be used in chlorophyll removal from vegetable oils. Chlorophyllase genes from Oscillatoria acuminata (OscChlase) and Citrus aurantium (CitChlase) were isolated, cloned, and expressed in E. coli. Bioinformatics analysis showed that both chlorophyllases shared a conserved GHSXG lipase motif responsible for their catalytic activity. SDS‐PAGE and immunoblot assays revealed that both enzymes had a molecular weight of 35 kDa. The purified chlorophyllases were stable at a broad range of temperatures and showed the highest activity at 40 °C. OscChlase and CitChlase exhibited the highest activity at pH 6.0 and 7.0, respectively. Enzyme kinetics analysis revealed that OscChlase was able to hydrolyze bacteriochlorophyll‐a more efficiently than the recombinant CitChlase (Vmax/Km of 0.38 for OscChlase vs. 0.01 min−1 mg protein for CitChlase). Instead, CitChlase hydrolyzed chlorophyll‐b more efficiently than OscChlase. Both enzymes were able to reduce the chlorophyll content of olive (from 623.1 to as low as 87.2 mg per kg oil) and canola oil (from 537.2 to as low as 101.1 mg per kg oil). The ratio of oil to the aqueous reaction media affected chlorophyll hydrolysis (P < 0.05). The lower the oil ratio was (10%), the higher the chlorophyll removal was (75–86%). The efficiency of CitChlase in chlorophyll removal was higher than that of OscChlase at oil ratios of 10 and 20, but lower at 30% ratio (P < 0.05). This is the first report on the application of recombinant OscChlase and CitChlase in chlorophyll removal (up to 86%) from vegetable oils.

Molecular, structural and biochemical characterization of a novel recombinant chlorophyllase from cyanobacterium Oscillatoria acuminata PCC 6304

Background Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacterium Oscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3).ResultsThe putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258.ConclusionsThe high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.

Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal

Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable. We found that phytenal accumulates during chlorotic stresses, for example, salt stress, dark-induced senescence, and nitrogen deprivation. The increase in the phytenal content is mediated at least in part independently of enzyme activities, and it is independent of light. Characterization of phytenal accumulation in the pao1 mutant affected in chlorophyll degradation revealed that phytenal is an authentic phytol metabolite derived from chlorophyll breakdown. The increase in phytenal was even stronger in mutants affected in the production of other phytol metabolites including vte5-2 (tocopherol deficient) and pes1 pes2 (fatty acid phytyl ester deficient). Therefore, phytenal accumulation is controlled by competing, alternative pathways of phosphorylation (leading to tocopherol production) or esterification (fatty acid phytyl ester production). As a consequence, the content of phytenal is maintained at low levels, presumably to minimize its toxic effects caused by its highly reactive aldehyde group that can form covalent bonds with and inactivate the amino groups of proteins.

Physiological and molecular analyses of chlorophyllase in sweet potatoes with different-colored leaves

Chlorophyllase (Chlase) is a hydrophobic enzyme that catalyzes the hydrolysis of chlorophyll (Chl) to chlorophyllide (Chlide) and phytol. The objectives of this research were to study Chlase characteristics of sweet potatoes (Ipomoea batatas, Ib) with various leaf colors: yellow (CN1927), green (TN57), and purple (CYY8467). Large variations in Chl and anthocyanin (Ant) and carotenoid (Car) existed in all plants, and CYY8467 contained significantly higher Chl, Ant, and Car levels compared to TN57 and CN1927. Various plants exhibited different Chlase activities, and Chl a degradation by Chlase of CYY8467 was higher than those of TN57 and CN1927. Nevertheless, Chlase activity in CN1927 exhibited a higher substrate preference toward Chl b than Chl a. Expression of the Chlase gene was detected in all leaves; however, IbChlase mRNA was most strongly active and regulated in TN57. The open reading frame sequences of IbChlase in TN57, CN1927, and CYY8467 shared 95% identity, and an eight-nucleotide sequence of Chlase in CYY8467 was missing due to a frame-shift mutation of the Chlase gene leading to early termination of gene translation. A phylogenetic analysis of Chlase indicated that 30 plants diverged into three clades, and sequences from TN57 and CN1927 were clustered together in a clade of the Chlase2.

Plants water soluble chlorophyll binding proteins act as enzyme-inhibitor pair

The hydrophilic water-soluble chlorophyll binding proteins (WSCP) which form complex with chlorophyll molecules have been numerously isolated from the chloroplasts of plants. Although, their molecular properties have been partly characterized, but their physio-biochemical roles are still unclear in the photosynthesizing organs. In this study, using bioinformatic tools WSCP pair were predicted to act as hydrolase and hydrolase inhibitor towards chlorophyll molecules. To enhance our information regarding the possible functions of WSCP, we cloned WSCP1 and WSCP2 cDNAs from Chenopodium album L. and Brassica oleracea L. leaves and expressed them as soluble maltose-binding fusion proteins in Escherichia coli. The purified fused products were subjected to chlorophyll hydrolyzing activity in vitro. The results showed that WSCP1 and WCSP2 are antagonistically involved in chlorophyll breakdown, while WSCP1 acts as chlorophyll hydrolyzing enzyme (with the hydrolysis rate of about 40% per 12 h), WSCP2 exerts inhibitory activity (with the inhibition rate of about 38% per 12 h) towards chlorophyll hydrolysis. This is the first ever time report speculates the hydrolase/inhibitory roles for WSCP and proposes that the relative activity of WSCP pair might balance and regulate the chlorophyll breakdown process in the photosynthetic apparatus of plants. It may open the new gate to investigate the potent roles of WSCP in plant system.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3–5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50–60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives.

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.

An integrated protein chemistry laboratory

Chlorophyll, the most abundant pigment in nature, is degraded during normal plant growth, when leaves change color, and at specific developmental stages. Chlorophyllase catalyzes the first chemical reaction in this process, that is, the hydrolysis of chlorophyll into chlorophyllide. Here, we describe a series of laboratory sessions designed to illustrate a sequence of experiments used as part of the scientific research process and to convey key biochemical concepts. The format guides students through the process of biochemical protein analysis, starting from a recombinant protein expression vector and working through a kinetic analysis of the purified protein. Over the course of these experiments, students learn protocols in basic protein chemistry that allow them to design and conduct a related experiment of their own interest. The described set of laboratories can be tailored to fit either a 4- or an 8-week series of experiments for use in either introductory or advanced biochemistry laboratory courses, respectively.

The effect of heavy metals accumulation on the chlorophyll concentration of Typha latifolia plants, growing in a substrate containing sewage sludge compost and watered with metaliferus water

Typha latifolia plants, commonly known as cattails, were grown in a mixture of sewage sludge compost, commercial compost and perlite. Four groups (A, B, C and D) were irrigated (once every 2 weeks) with a solution containing different concentrations of Cd, Cu, Ni, Pb and Zn, where in the fifth (group M) tap water was used. At the end of the 10 weeks experimental period the mean concentration of Ni, Cu and Zn in the roots and leaves of the plants in the four groups was significantly larger to that of the plants of group M. A linear regression test satisfactorily correlated the metals’ concentrations in the irrigation solutions with the metals concentration in the leaves and roots of groups A, B, C and D. The concentration of total chlorophyll, chlorophyll a (chla) and chlorophyll b (chlb) in the leaves of the developing plants was also monitored in 2 weeks intervals. Groups A, B, C and M presented an increasing concentration of total chlorophyll, with time. In group D (stronger solution), the mean total chlorophyll concentration was reduced from 1080.69 μg/g fresh weight (f.w.) in the 8th week to 715.14 μg/g f.w., in the 10th week, a probable evidence of inhibition. When statistically tested, it was suggested that there was no significant difference between the mean chlorophyll values of the groups in each set of samples, concluding that no significant toxic action was imposed in the plants by the metals. However, when similar statistical analysis was implemented in the ratios of chla and chlb, there was significant reduction of the ratios in groups D plants, suggesting some increase in chlorophyll hydrolysis due to the metals accumulation (toxic effect) in comparison with the other groups.

In vivo effects of chlorophyllin on the antitumour agent cyclophosphamide

Cyclophosphamide (CP) is a potent antitumour agent used against many forms of cancer and against certain other diseases. Chlorophyllin (CHL), which is obtained by hydrolysis of chlorophyll to remove phytyl alcohol, is an efficient antimutagenic agent and has been used as a dietary supplement or to diminish the intensity of the discomforting side effects of CP therapy. We undertook to determine the antimutagenic effectiveness of CHL against CP in a mouse model and to determine whether the antitumour efficacy of CP was compromised in vivo by CHL treatment. Experiments utilised CHL administered either in drinking water (1%) for 2 days before treatment, or by gavage (200 mg/kg) 2 hr before treatment with CP (220 mg/kg). Urinary mutagenicity following CP treatment, as determined by the Salmonella/microsome assay, was decreased by both regimes of CHL co-treatment. Similarly, the increase in micronuclei in bone marrow polychromatic erythrocytes in response to CP was reduced by concomitant CHL treatment. In contrast, antitumour efficacy, as determined by growth delay of Colon 38 adenocarcinomas, was not diminished by CHL treatment. We conclude that CHL may have beneficial effects when used in combination with CP therapy. © 1997 Wiley-Liss, Inc.

In vivo effects of chlorophyllin on the antitumour agent cyclophosphamide.

Cyclophosphamide (CP) is a potent antitumour agent used against many forms of cancer and against certain other diseases. Chlorophyllin (CHL), which is obtained by hydrolysis of chlorophyll to remove phytyl alcohol, is an efficient antimutagenic agent and has been used as a dietary supplement or to diminish the intensity of the discomforting side effects of CP therapy. We undertook to determine the antimutagenic effectiveness of CHL against CP in a mouse model and to determine whether the antitumour efficacy of CP was compromised in vivo by CHL treatment. Experiments utilised CHL administered either in drinking water (1%) for 2 days before treatment, or by gavage (200 mg/kg) 2 hr before treatment with CP (220 mg/kg). Urinary mutagenicity following CP treatment, as determined by the Salmonella/microsome assay, was decreased by both regimes of CHL co-treatment. Similarly, the increase in micronuclei in bone marrow polychromatic erythrocytes in response to CP was reduced by concomitant CHL treatment. In contrast, antitumour efficacy, as determined by growth delay of Colon 38 adenocarcinomas, was not diminished by CHL treatment. We conclude that CHL may have beneficial effects when used in combination with CP therapy.

BIOCATALYSIS OF CHLOROPHYLLASE IN CANOLA OIL USING ORGANIC SOLVENT SYSTEMS

The hydrolytic activity of a partially purified chlorophyllase, obtained from an algal source, was investigated in a refined-bleached-deodorized (RBD) canola oil model system using various organic solvent media, including water/miscible-organic, biphasic organic, and micellar ternary systems. Although the use of a a ternary micellar system containing Span 85 was found to be the most appropriate medium for the hydrolysis of chlorophyll, the hydrolytic activity of chlorophyllase declined as oil content increased. The effects of various parameters, including surfactant concentration, hexane/buffer proportion, enzyme content, reaction time, incubation temperature, shaker speed, and activator concentration, on chlorophyllase activity in the micellar ternary systems containing 20% RBD oil were also investigated. In addition, phytol displayed a noncompetitive inhibition in reaction media containing polysorbate 80 and Span 85. The plot of 1/v versus oil concentrations revealed that the oil had K i values of3. 73 and 4.56% in the micellar systems containing polysorbate 80 and Span 85, respectively. Moreover, the presence of 10% oil in the micellar system containing span 85 and polysorbate 80 decreased V max values of chlorophyllase activity by 6.2 and 9.6-fold, respectively.

Pigment removal from canola oil using chlorophyllase

AbstractFrost‐damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 µg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water‐soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone‐dried chloroplasts) from 15 to 20‐day‐old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X‐100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg2+ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15µM or 13.5 µg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr.With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (Ki=11 µM or 3.3 µg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity.Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).

Interactions between cholorophyllase, chlorophyll a, plant lipids and Mg 2+

Chlorophyllase-catalyzed conversion of purified chlorophyll a does not occur, or occurs only slightly, in the absence of lipids. The enzyme reaction is shown to be activated by mixed spinach chloroplast lipids and also, in varying degrees, by several single plant lipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidylglycerol and, to a lesser extent, phophatidylcholine and sulphoquinovosyl diacylglycerol). β-Carotene, in the absence of other lipids, inhibits chlorophyllase-catalyzed chlorophyll a conversion. Interaction of various plant lipids with chlorophyll in an aqueous medium has been studied by absorption and fluorescence spectroscopy. Digalactosyl diagylglycerol, phosphatidylglycerol and sulphoquinovosyl diacylglycerol stimulate the formation of crystalline chlorophyll a. In the presence of monogalactosyl diacylglycerol in its hexagonal phase chlorophyll a is monomerized, and the chlorophyll molecules become fluorescent. Chlorophyllase (EC 3.1.1.14) is shown to combine with monogalactosyl diacylglycerol and with β-carotene. Evidence is presented that small quantitaties of these and other lipids may stabilize the enzyme conformation. Cations, such as Mg 2+, stimulate the formation of chlorophyll aggregates in an aqueous medium. Nevertheless, in combination with dithiothreitol, they can still activate chlorophyllase-catalyzed chlorophyll hydrolysis. This activation is shown to occur only in the presence of lipids. It is suggested that the positive effect that cations have on the enzyme reaction is due, at least in part, to their interaction with the headgroups of chlorophyllase-associated lipids. The role that lipids and cations may play in the modulation of chlorophyllase activity in natural membranes is discussed.

Studies on chlorophyllase. The mechanism of the action of lecithin liposomes on enzyme activity and the function of the carbohydrate moiety of the enzyme

A sensitive and continuous fluorescence assay of chlorophyllide formation in the presence of lecithin liposomes has been developed. The mechanism of the enhancing effect of lecithin on chlorophyllase-catalyzed hydrolysis of chlorophyll has been elucidated. Using both fluorescence and biochemical assays, the function of the concanavalin A-reactive carbohydrate moiety of chlorophyllase has been investigated. Experiments on the interaction of chlorophyllase with concanavalin A show that the sugar group not only stabilizes the enzyme, but also is essential for the manifestation of enzyme activity. From a comparison of the results obtained with solubilized and membrane-bound enzyme, it is concluded that the active site is situated on the outside of the thylakoid membrane. Mg 2+, in combination with dithiothreitol, activates chlorophyllase. In the presence of Mg 2+, an abnormal pH dependence of the inhibiting effect of concanavalin A on chlorophyllase-catalyzed chlorophyll hydrolysis and the reversibility of this effect through the addition of α-methyl-D-mannoside have been observed. The cause of this atypical reaction as well as of other Mg 2+ effects is discussed.

Kinetics and mechanism of the hydrolysis of chlorophyll a in ternary solvent microemulsion media

Kinetics and mechanism of the hydrolysis of chlorophyll a in ternary solvent microemulsion media

Influence of lecithin liposomes on chlorophyllase-catalyzed chlorophyll hydrolysis. Comparison of intramembraneous and solubilized Phaeodactylum chlorophyllase

1. 1. Chlorophyllase-catalyzed chlorophyll hydrolysis is greatly enhanced by the addition of divalent cations (Mg 2+) combined with a reducing agent (dithiothreitol, ascorbate). A similar effect is obtained by the addition of lecithin. In the presence of lecithin, dithiothreitol has only slight or no influence on chlorophyll hydrolysis. Mg 2+ eliminates the activating effect of lecithin. 2. 2. In the absence of Mg 2+ + dithiothreitol or of lecithin, Triton X-100 has a slight activating effect on chlorophyllase-catalyzed chlorophyll hydrolysis, but only at low concentrations (0.01–0.02%). In the presence of Mg 2+ and dithiothreitol or of lecithin, Triton X-100 (greater than or equal to 0.02%) inhibits this reaction. 3. 3. Whereas chlorophyllase combines with chlorophyll, no combination of chlorophyllase and lecithin could be detected. 4. 4. Solubilized chlorophyllase is stabilized by its substrate, chlorophyll. Enzyme stabilization is eliminated by lecithin, whereas in the absence of chlorophyll, denaturation is somewhat increased by dithiothreitol. 5. 5. No clear difference was found between the actions of intramembraneous and solubilized chlorophyllase. The results suggest that chlorophyllase is situated within membranes in such a way that the active group protrudes into the aqueous medium surrounding the membrane. 6. 6. A hypothesis explaining the activating effects which Mg 2+ combined with a reducing agent and lecithin have upon chlorophyllase-catalyzed chlorophyll hydrolysis is presented.

The Effect of Hydrogen Ion Concentrations in Simulated Rain on the Moss Tortula ruralis (Hedw.) Sm.

Gametophores of the moss Tortula ruralis were treated with sulfuric acid solutions at pH values of 1.0 to 6.0. The concentration of chlorophylls a+b decreased between pH6 and pH3 followed by a more rapid decline between pH3 and pH1. Chlorophyll a was more sensitive to acid than chlorophyll b. Oxygen production based on chlorophyll was relatively unaffected between pH6 and pH2, whereas photosynthesis based on dry weight decreased uniformly in relation to decrease in pH. Respiration was insensitive to pH values as low as pH2. Simulated acid rain appeared to effect a reduction in photosynthesis largely through acid hydrolysis of chlorophyll a.