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Three-Dimensional in the Inner Seed Naoto
Chromatographic Coats of Pumpkin
Department of Chemistry, Faculty of Science and Technology, Kinki Kowakae, Higashiosaka 577, Japan
vinyl-protopheophytin esterifying ester
seed coats Three
a, were moiety,
of precursors, Each
The inner seed coats of pumpkin is known to have an abundance of protochlorophyllsl-3, and their isolation and characterization from Cucurbita maxima Duch were reported recently.4 Several compounds were separated by high-performance liquid chromatography (HPLC), and three were identified by absorption spectrometry and fast atom bombardment mass spectrometry (FAB/ MS). For such studies, a simple and rapid method was required for the separation and identification of such structurally similar compounds. An effective method for characterizing these precursors was found in threedimensional high-performance liquid chromatography (3D-HPLC). Many precursors of chlorophyll could be identified from one three-dimensional chromatogram. Reported here are the results of application of this method to the study of inner seed coat protochlorophylls of pumpkin.
tetrahydrogeranyl a was
a and 2variety
The phytol on the three-
two types of mixed solutions (acetonitrile : acetone= 55 : 45, v/ v and acetonitrile :1-propanol : water=50 :45 : 5, v/ v/ v) at 18°C. The flow rates were 1.5 and 1.0 cm3/ min, respectively. All chemicals used were of reagent grade, and the experiments were undertaken under dark green light to avoid photolysis of the precursors.
Figure 1 shows the three-dimensional chromatogram of pigments extracted from inner seed coats. The elution bands were recorded as a function of x, y and z : x is elution time, y is absorbance and z is wavelength. The pigments were eluted with a mixture of acetone : acetonitrile =55 : 45, v/ v, at a flow rate of 1.5 cm3/ min at 18°C. Many peaks could be identified as precursors of chlorophyll; the absorption peak in the wavelength
Pumpkin seeds (10 - 20 pieces) were soaked in water for 1 or 2 h. The softened wet seed coats were cut, and each dark green inner seed coat was removed. Pigments were extracted from these coats with acetone (about 50 ml). The extract was filtered through anhydrous sodium sulfate layer. The filtrate was fractionated by 3D-HPLC with a Model LC-6AD system (Shimadzu) attached to a Dupont Zorbax ODS column (4.6X250 mm) and subjected to detection using a photo-diode array spectrophotometer, Model SPD-M6A (Shimadzu). A tungsten lamp and a D2-lamp were used for the wavelength regions from 380 to 670 nm and from 200 to 500 nm, respectively. The pigments were eluted with
region 420 to 440 nm corresponds to the B-band of porphyrin and absorption maxima at 500 to 700 nm region to the Q-band. From these peaks, it became clear that the extracted pigments of the inner seed coats of pumpkin contain more than ten protochlorophyll species. Figure 2 shows the two-dimensional chromatogram, which is the projection plane of Fig. 1 from the upper face. The wavelength is recorded on the vertical axis and the retention time horizontally, with the peak height being shown by a contour line. There are broad unresolved peaks overlapping each other in the range of 2 to 5 min. After the elution, we achieved good separation of the major peaks, which we numbered in the elution order. Peaks 1- 3 had absorption maxima at 440 nm, and peaks 4 - 9 had them at nearly 420 nm. From this difference in the absorption maximum, these peaks were divided into two groups. This classification was confirmed from the HPLC chromatogram of Fig. 3, which shows two spectra detected at 440 nm and 420 nm,
seed coat extracts.
respectively. Peaks 1, 2 and 3 have higher absorbances at 440 nm than those at 420 nm. On the other hand, peaks 4 to 10 have higher absorbances at 420 nm than those at 440 nm. The fractions of ten peaks were taken in a fraction collector, and their absorption spectra were measured in the visible region. Fractions corresponding to peaks 1, 2 and 3 show the same absorption spectrum, which has an absorption maximum at 437 nm for B-band and at 574 nm and 623 nm for Q-band, as shown in Fig. 4. This spectrum agrees with that of 2,4divinyl-protochlorophyll a (divinyl-protochlorophyll a) as can be seen from Table 1. Therefore, the chromatographic separation of the three peaks can be attributed to the difference in the structure of alcohol moieties esterified to the propionic acid of divinylprotochlorophyllide a. The three alchohols shown in Fig. 5 are geranyl geraniol (GG), dihydrogeranyl geraniol (DHGG) and tetrahydrogeranyl geraniol (THGG) which are all precursors of phytol in its biosynthesis. The structural differences have been confirmed by previous FAB/ MS results. From the elution order, peak 1 was identified as divinyl-protochlorophyll a (4V-Pchl. a) esterified with GG and peaks 2 and 3 as those of DHGG and THGG, respectively. Also, the peaks of 5, 7, 9 and 10 showed the same absorption maxima at 421 nm for the B-band and at 528 nm, 567 nm, 589 nm and 642 nm for the Qband of oxorhodo-type.ll These absorption spectra agree with that of divinyl-protopheophytin a (4V-Ppheo. a). Therefore, these species of peaks 5, 7, 9 and 10 were identified as divinyl-protopheophytin a-GG, -DHGG, -THGG and -phytol esters , respectively. The peaks 4, 6 and 8 have the same absorption maxima at 417 nm for the B-band and at 528 nm, 567 nm, 588 nm and 639 nm for the Q-band of oxorhodo-type. This absorption spectrum agreed with that of monovinyl-protopheophytin a (Ppheo. a), showing peaks 4, 6 and 8 to be monovinylprotopheophytin a-GG, -DHGG and -THGG esters, respectively.
There was the possibility of protochlorophyll, that
protochlorophyll nation. This
of unfavorable demetalation is, the transformation from
to protopheophytin, during problem was examined from
points. One was The acetone extract
a; R,: 2,4-divinyl-protochlorophyll
the examitwo view-
the photolysis of protochlorophyll. was exposed to light for at least 10 h,
but no increase in the heights of peaks 4, 5, 6, 7, 8, 9 and 10 was observed, although the heights of the broad unresolved peaks at the head of the chromatogram tended to increase. The other was the effect of acidity of the residual silanol of ODS, because demetalation of protochlorophyll was frequently observed on the ODSthin layer chromatography. However, previously permeated acetonitrile, which is a mild basic solvent, could protect protochlorophyll from demetalation. Although it is difficult to rule out demetalation completely, these results show that two groups of protopheophytin exist, divinyl-protopheophytins and monovinyl-protopheophytins. The heights of the peaks show that the former are the dominant precursors. The only precursor esterified with phytol was that of peak 10, divinyl-protopheophytin a-phytol ester. However, there was the possibility of another precursor esterified with phytol. Study of the phytol substance revealed, as shown in Fig. 6, that linear relationships exist between the logarithmic capacity factors (k') of three groups and the number of double bonds of alcohol attached to the propionic carboxyl group. The three groups were the divinyl-protopheophytin a group, the monovinyl-protopheophytin a group and the divinylchlorophyll a group. These linear relations indicate that the retention time of substances increases with a decrease in the number of double bonds, that is, the plots of the substances esterified with phytol which has one double bond should be placed at the upper most point on each line, just as point 10 in Fig. 6. Phytol esters of monovinyl-protopheophytin a and divinyl-protochlorophyll a, which are not detected on the chromatogram, can be placed at points 0 and Q, respectively. Both marked points can overlap with the points of number 9 and 5, that is, the weak peaks of phytol esters of monovinylprotopheophytin a and divinyl-protochlorophyll a may overlap with peaks of other precursors.
and log k' on
To separate the broad unresolved peaks at the head of the chromatogram, the eluting solvent was changed to the mixture of acetonitrile :1-propanol : water (50 : 45 : 5, v/v). These peak heights tended to increase with light irradiation. The contour chromatogram (Fig. 7) shows good separation at the front part in the range of 2 to 8 min, but poorer separation in the back part. The ultraviolet region has peaks of some species other than porphyrins, which were presumed to be from some proteins and lipids contained in the inner seed coats of pumpkin. Figure 8 is an enlarged view of Fig. 7 in the region of 2 to 8 min. There are six peaks which have absorption maxima in the region of 420 to 440 nm, the wavelength region agreeing with the B-band of porphyrin. These six peaks can be divided into two groups, based on the difference in the maximum wavelength of the peak. One group has its maximum wavelength at 437 nm, which corresponds to the B-band of divinylprotochlorophyll a, and the other group at 421 nm, which corresponds to the B-band of divinyl-protopheophytin a. These three peaks of both groups display the same elution behavior, that is, they are eluted at approximately regular intervals, and the peak height increases with the elution time. These elution intervals and peak height pattern are also identical with those of the divinyl-proto-
Fig. 6 Relationship between main protochlorophylls.
phenomenon indicates that the presence of oxygen may induce addition of oxygen to protochlorophyll a and protopheophytin a and result in an increase of molecular polarity. However, the addition of oxygen is difficult to detect from the absorption spectra. Identification of the highly polar compounds by FAB/ MS is now underway. Financial support from the Ministry of Education, Science and Culture and from Kinki University is gratefully acknowledged. The authors express their sincere thanks to Mr. Tomio Fujita, Shimadzu Corporation (HPLC Kyoto Analytical Applications Center) for his useful help and discussion.
References Fig. on
polar protopheophytin chlorophyll a.
protochlorophylls. a; X-Pchl.
and a: polar
log k' highly proto-
chlorophyll a and divinyl-protopheophytin a esterified with GG, DHGG and THGG. Figure 9 shows the linear relationships in the plots of log k' of quasi-protochlorophylls a (X-Pchls. a) and quasi-protopheophytins a (X-Ppheos. a) versus the numbers of double bonds of alcohol. They are the same as those of protochlorophylls a and protopheophytins a. Thus, these two groups seem to be esterified with GG, DHGG and THGG, respectively. However, the quasisubstances have shorter elution times than those of protochlorophylls a and protopheophytins a, indicating that they are more polar species. In this case, absorption spectrometry cannot distinguish between quasi-protochlorophyll and protochlorophyll, although the polarities of these molecules differ significantly. The fluorescence of chlorophyll is quenched due to the oxygen-adduct to the porphyrin nucleous.12 This
1. C. Houssier and K. Sauer, Biochem.Biophys. Acta, 172, 476 (1969). 2. R. K. Ellsworth and C. A. Nowak, Anal. Biochem., 57, 534 (1974). 3. Y. Shioi and T. Sasa, Plant Cell PhysioL, 23,1315 (1982). 4. N. Mukaida and Y. Nishikawa, Nippon Kagaku Kaishi, 1990, 1244. 5. 0. T. G. Jones, Biochem. J., 101, 153 (1966). 6. 0. T. G. Jones, Biochem. J., 96, 6P (1965). 7. V. M. Koski and J. H. C. Smith, J. Am. Chem. Soc., 70, 3558 (1948). 8. C. S. French, in "Handbuch der Pflanzenphyiolosie vol.5", ed. W. Ruhland, Part 1, p. 252, Springer, Berlin, 1960. 9. R. V. Stanier and J. H. C. Smith, Carnegie Inst. Wash. Yearbook, 58, 336 (1959). 10. 0. T. G. Jones, Biochem. J., 88, 335 (1963). 11. K. M. Smith, "Porphyrins and Metalloporphyrins", p. 21, Elsevier Scientific Publishing Company, Amsterdam, 1975. 12. Y. Yoshitake, M. Tanino, K. Morishige, T. Shigematu and Y. Nishikawa, Bunseki Kagaku, 38, 182 (1989). (Received March 11, 1993) (Accepted July 6, 1993)