Tsutomu Sasa

Chlorophyll analysis by high-performance liquid chromatography

Abstract The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.

Purification by affinity chromatography and properties of uroporphyrinogen I synthetase from Chlorella regularis

Uroporphyrinogen I synthetase (porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8) from Chlorella regularis was purified to homogeneity by affinity chromatography on porphobilinogen-AH-Sepharose 4B, which was prepared by reacting carbodiimide with substrate, porphobilinogen. The enzyme was purified 232-fold from the initial crude extract and specific activity was 348 nmol porphyrinogen I formed (mg protein)-1 . h-1 at pH 7.4. The molecular weight of the enzyme was 35 000-36 000 as determined by Sephadex G-100 gel filtration. This enzyme was acidic protein having an isoelectric point of 4.2. The enzyme exhibited a single pH optimum at a pH value of 7.4 both in phosphate and Tris-HCl buffer. The Km value for porphobilinogen was 89 microM as measured by its consumption and 85 microM when uroporphyrin formation was used. The Arrhenius plot obtained from the enzyme activity measurements appeared triphasic with breaks occurring at 35 and 46 degrees C and activation energy was calculated to be 21 700 (10-35 degrees C), 12 700 (35-46 degrees C) and 1800 cal . mol-1 (46-65 degrees C). This enzyme was heat stable and the enzyme still retained 87% of activity, even after 1 h incubation at 75 degrees C.

Separation of non-esterified chlorophyls by ion-suppression high-performance liquid chromatography

Chlorophyllides and pheophorbides were separated by ion-suppression high-performance liquid chromatography using an octadecyl silica column eluted with 80–95% (v/v) methanol in water containing 13 mM acetic (final pH 4.2) as the suppessing ion. The separated pigments were detected fluorometrically with a sensitivity in the picomol range without artifact formation. This technique can be used not only for the determination of non-esterified chlorophylls, but also for esterified chlorophylls, thus enabling the simultaneous identification of chlorophylls and their derivatives.

Immobilization of photochemically-active chloroplasts onto diethylaminoethyl-cellulose

The use of immobilized photosynthetic systems as multifunctional biocatalysts is a novel approach to the problem of photobiological energy conversion. The main advantages in the use of the immobilized photosystems are the increased stability and the flexibility in reactor design. Recently, reports have appeared which emphasize the increasing importance of research in the field [l-5]. Chloroplasts have been immobilized by entrapment within polyacrylamide gel [ 11, polyvinyl alcohol [2] and microcapsules [3]. However, high retention of photosynthetic activity was not achieved, in contrast to bacterial chromatophores immobilized by entrapment within polyacrylamide gel [4], because of the instability of photosystem II (PS II) activity of chloroplasts. Enzyme immobilization by adsorption is simple, mild and reversible permitting reuse of both enzyme and the substratdm. Thus, adsorption may be a suitable method for immobilization of chloroplasts. Diethylaminoethyl (DEAE)-cellulose, one of the anion exchangers, is commercially available and widely used as an ion exchanger for protein fractionation. It has already been used effectively as a substratum for various enzymes such as acid acylase [6], glucoamylase [7], glucose isomerase [8] and invertase [9]. We have investigated its application for preparation of immobilized chloroplasts with a high retention of photochemical activities. Here we describe the preparation and properties of chloroplasts immobilized by adsorption to DEAEcellulose. Some observations on its use in the contin-

[50] Purification of solubilized chlorophyllase from Chlorella protothecoides

Publisher Summary This chapter describes a simple purification procedure for the preparation of solubilized chlorophyllase from Chlorella protothecoides . Chlorophyllase activity is conveniently determined by the rate of chlorophyllide a formation. This method was based on the partition of chlorophyll and chlorophyllide between n -hexane and aqueous acetone. The increase in formed product, chlorophyllide a concentration in the aqueous acetone layer is measured spectrophotometrically. Chlorophyllase activity is also assayed by the alcohol transesterifying activity. Alcohol transesterifying activity is determined by the use of methanol as an alcohol and by measuring the formation of methylchlorophyllide. In this reaction, chlorophyllide formation is negligible and methyichlorophyllide can be identified and quantified by thin-layer chromatography. The steps of purification are carried out at 0–4°; and include butanol solubilization, ammonium sulfate fractionation, first Sephadex G-200 gel filtration, sepharose CL-6B gel filtration, and second Sephadex G-200 gel filtration of the enzyme. Optimal acetone concentration for the assay of chlorophyllase depends on the degree of purification. The pH of all reaction mixtures should be adjusted after addition of acetone. Chlorophyllase prepared by the method described is homogeneous (around peak fractions) as determined by polyacrylamide gel electrophoresis. Chlorella enzyme is an acidic protein and does not require metal ion and thiol compound for activity. This enzyme shows high-molecularweight aggregates similar to other enzymes. Kinetic studies with purified enzyme indicated that Chlorella enzyme consists of at least two enzymes. One enzyme catalyzes hydrolysis of chlorophylls and the other, alcohol transesterification of chlorophylls and its reverse reaction. Other physicochemical and kinetical properties of the Chlorella enzyme and the purified enzyme from various sources are tabulated in the chapter.

Esterification of chlorophyllide b in higher plants

Abstract Chlorophyllide b and four chemically different chlorophyll b specieis, chlorophyllide b esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol have been detected in addition to the same derivatives of chlorophyll a in the greening cotyledons of cucumber. These esters could be separated and determined by high-performance liquid chromatography. The results suggest that chlorophyll b phytol is formed from the esterification of chlorophyllide b and geranylgeraniol followed by three hydrogenations of the alcohol moiety, as in the case of chlorophyll a and protochlorophyll phytol formation

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides.

Abstract A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)−1 min−1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking.

Purification and properties of l-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.

Analysis of porphyrin precursors, 5-aminolevulinic acid derivatives by isotachophoresis

Porphyrin precursors, 5-aminolevulinic acid (ALA), 4,5-dioxovaleric acid (DOVA), and porphobilinogen (PBG) can be simultaneously and conveniently analyzed by isotachophoresis. The quantitative analysis requires a minimum of 12 nmol for ALA, 4 nmol for DOVA, and 35 nmol for PBG. The reproducibility determined as coefficient of variation for qualitative and quantitative determination is within 4 and 5%, respectively. Isotachophoresis permits simple, efficient and quantitative detection, and identification of ALA derivatives in a mixture.