Spectrofluorometric estimation of intermediatesof chlorophyll biosynthesis: Protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide

ANALYTICALBIOCHEMISTRY 2 0 6 , 125-130 (1992)

Spectrofluorometric Estimation of Intermediates of Chlorophyll Biosynthesis: Protoporphyrin IX, Mg-Protoporphyrin, and Protochlorophyllide 1 P i n k i H u k m a n i a n d B a i s h n a b C. T r i p a t h y 2 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Received June 3, 1992

A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fuorescence emission spectra is d e v e l o p e d . At r o o m t e m p e r a t u r e ( 2 9 3 ° K ) p r o t o p o r p h y r i n I X is m o n i t o r e d f r o m i t s e m i s s i o n m a x i m u m , 6 3 3 n m , w h e n e x c i t e d at 4 0 0 n m (E4oo/r633). P r o t o c h l o r o p h y l l i d e is e s t i m a t e d at 6 3 8 n m , w h i l e b e i n g e x c i t e d at 4 4 0 n m (E440/Fe3s). M g - p r o t o p o r p h y r i n + M g - p r o t o porphyrin monoester pool has emission around 589592 nm. Therefore the integration value of the emiss i o n b a n d t h a t e x t e n d s f r o m 5 8 0 to 6 1 0 n m is t a k e n to c a l i b r a t e its c o n c e n t r a t i o n . T h i s s p e c t r o f l u o r o m e t r i c m e t h o d d e s i g n e d for t h e d e t e r m i n a t i o n o f p r o t o p o r p h y r i n IX, e s t e r i f i e d a n d n o n e s t e r i f i e d M g - p r o t o p o r p h y r i n pool, a n d p r o t o c h l o r o p h y l l i d e is f a r s u p e r i o r t o a v a i l a b l e s p e c t r o p h o t o m e t r i c m e t h o d s a n d e s t i m a t e s as l o w as 1 nM c o n c e n t r a t i o n o f p l a n t p i g m e n t s . A s m i n u t e quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates a n a l y t i c a l u n c e r t a i n t i e s d u e to r e c o v e r y l o s s e s c a u s e d by chromatography. However, only dilute samples can b e e s t i m a t e d b y t h i s s p e c t r o f l u o r o m e t r i c m e t h o d as t h e quantitative relation between fluorescence and concent r a t i o n d e v i a t e s f r o m l i n e a r i t y at high, i.e., a b o v e 1 5 0 riM, c o n c e n t r a t i o n s o f p i g m e n t to be q u a n t i f i e d . © 1992 Academic Press, Inc.

The chlorophyll (Chl) 3 molecules are synthesized from 5-aminolevulinic acid (ALA) via various interme-

1 This work was supported by Grant DST/SP/SO/A44-88 from the Department of Science and Technology, Government of India. 2 To whom correspondence should be addressed. 3 Abbreviations used: ALA, 5-aminolevulinic acid; Chl, chlorophyll; Chlide, chlorophyllide; HEAR, Hexane-extracted acetone residue 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

diates (1). Two ALA molecules condense to form one porphobilinogen (PBG) unit, which is the first pyrrolic intermediate in the pathway. Four PBG units are linked together to produce a linear tetrapyrrole which is unstable. The latter is closed to form the first cyclic tetrapyrrole, uroporphyrinogen III, where ring D is inversed. The conversion of uroporphyrinogen III to coproporphyrinogen III is carried out by decarboxylation of four acetic acid residues to methyl groups. Coproporphyrinogen III is converted to protoporphyrinogen IX by decarboxylation of propionic acid residues at 2 and 4 positions to vinyl groups. Removal of six electrons from protophyrinogen macrocycle yields protoporphyrin IX (Proto IX), which is the precursor of Chl and cytochromes, etc. (2). The key branch point step to Chl is the insertion of Mg into protoporphyrin IX by Mg chelatase enzyme. Methylation of propionic acid residues at position 6 of Mg-protoporphyrin IX results in the formation of Mg-protoporphyrin monoester (MPE) (3). The conversion of M P E to protochlorophyllide (Pchlide) is accomplished by the joining of esterified propionate moiety at position 6 to the 3,-meso carbon of the porphyrin to form the isocylic pentanone ring, which is accomplished via/~-oxidation of the methyl propionate group followed by oxidative cyclization (4). Transformation of Pchlide to chlorophyllide (Chlide) is accomplished by the reduction of ring D. Pchlide is phototransformed to Chlide by Pchlide reductase enzyme (5). To elucidate the Chl biosynthetic pathways, to determine the effect of certain stresses, such as heat or clod, on Chl biosynthesis (6), or to monitor the a m o u n t of

solvent mixture; MPE, Mg-protoporphyrin monoester; MP(E), Mg protoporphyrin + Mg-protoporphyrin monoester; PBG, porphobilinogen; Pchlide, protochlorophyllide; Proto IX, protoporphyrin IX; Mg-proto, Mg-protoporphyrin. 125

126

HUKMANI AND TRIPATHY

o v e r a c c u m u l a t e d p o r p h y r i n s inducing p h o t o d y n a m i c d a m a g e to t h e p l a n t s (7-9), t h e various pools of interm e d i a t e s of t h e Chl b i o s y n t h e t i c p a t h w a y such as P r o t o IX, M P E , a n d Pchlide n e e d to be quantified. A s p e c t r o p h o t o m e t r i c m e t h o d for q u a n t i t a t i v e l y e s t i m a t i n g t h e s e p i g m e n t s f r o m t h e i r m i x t u r e s was developed (10). In s m a l l a m o u n t s of tissues a n d especially in isolated etiop l a s t s or c h l o r o p l a s t s t h e pools of the above i n t e r m e diates are too small, i.e., a few picomoles, to be estim a t e d s p e c t r o p h o t o m e t r i c a l l y . As P r o t o IX, M P E , a n d Pchlide fluoresce a n d s p e c t r o f l u o r o m e t r y is a v e r y sensitive tool, t h e fluorescence of t h e s e c o m p o u n d s c a n be m e a s u r e d for quantification. P r o t o I X a n d Pchlide h a v e o v e r l a p p i n g fluorescence spectra. T h e s e p i g m e n t s are m i x e d with Chlide, which also p a r t i t i o n s into h e x a n e e x t r a c t e d a c e t o n e phase. Chlides also fluoresce at 633 a n d 638 n m , t h e p e a k p o s i t i o n s of P r o t o I X a n d Pchlide, respectively. T h e r e f o r e fluorescence of P r o t o I X (E4o0 F633) a n d Pchlide (E440 F63s) n e e d s to be c o r r e c t e d for fluorescence due to Chlide a n d due to Pchlide or P r o t o IX, respectively, which are p r e s e n t in the h e x a n e - e x t r a c t e d a c e t o n e residue solvent mixture. T h e Chlide pool consists of b o t h Chlide a a n d Chlide b a n d t h e y c a n n o t be e s t i m a t e d f r o m t h e i r fluorescence e m i s s i o n spectra. H o w e v e r , t h e y could be e s t i m a t e d f r o m t h e i r fluorescence e x c i t a t i o n spectra. In the p r e s e n t investigation no a t t e m p t to q u a n t i t a t e Chlide a a n d Chlide b s p e c t r o f l u o r o m e t r i c a l l y is m a d e . In t h e h e x a n e - e x t r a c t e d a c e t o n e residue ( H E A R ) fraction of the a c e t o n e e x t r a c t of chloroplasts, Chlide a a n d Chlide b pools are p r e s e n t in sufficient q u a n t i t i e s a n d m a y be e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y . T h e M P ( E ) pool is heterogen o u s a n d consists of M g - p r o t o + M g - p r o t o m o n o e s t e r . B o t h p i g m e n t s are p a r t i t i o n e d into H E A R phase. M g p r o t o a n d M P E h a v e n e a r identical fluorescence spect r a l p r o p e r t i e s . T h e r e f o r e , M g - p r o t o a n d M P E are estim a t e d t o g e t h e r . T h e p r e s e n t investigation a i m s to q u a n t i t a t i v e l y e s t i m a t e P r o t o IX, M P ( E ) , a n d Pchlide, the t e t r a p y r r o l e i n t e r m e d i a t e s of Chl m e t a b o l i s m , b y spectrofluorometry. MATERIALS AND METHODS

Plant material. B a r l e y (Hordeum vulgare L.cv IB65) a n d c u c u m b e r (Cucumis sativus L.cv p o i n s e t t e ) p l a n t s were grown in t h e d a r k for 6 d a y s on g e r m i n a t i o n p a p e r . Source of Pchlide and MPE. T h e s e t e t r a p y r r o l e s were p r e p a r e d f r o m 6 - d a y - o l d etiolated b a r l e y leaves which were induced to a c c u m u l a t e m a s s i v e a m o u n t s of t h e s e c o m p o u n d s . T h i s was achieved by i n c u b a t i n g 10-g b a t c h e s of excised b a r l e y leaves in p e t r i dishes, 20 c m in d i a m e t e r a n d c o n t a i n i n g 25 ml of i n c u b a t i o n m e d i u m . T h e l a t t e r c o n s i s t e d of 4.5 mM A L A a n d 3.5 mM 2,2'dipyridyl (11,12). Pigment extraction. C u c u m b e r c o t y l e d o n s or b a r l e y leaves were e x t r a c t e d in 200 ml of acetone:0.1 N N H 4 O H

(9:1). T h e fully esterified t e t r a p y r r o l e s were e x t r a c t e d into h e x a n e while t h e m o n o - a n d dicarboxylic t e t r a p y r roles r e m a i n e d in the h e x a n e - e x t r a c t e d a c e t o n e residue fraction. T h e p i g m e n t s were e x t r a c t e d f r o m this H E A R fraction w i t h diethyl e t h e r a f t e r addition of 1/17 vol of s a t u r a t e d NaC1 a n d 1/70 vol of 0.5 M p h o s p h a t e buffer ( p H 7.0). Purification of monocarboxylic tetrapyrroles. T h e ether extracts containing the monocarboxylic tetrapyrroles were c o n c e n t r a t e d b y v a c u u m drying a n d dried u n d e r N2 gas a n d were c h r o m a t o g r a p h e d on t h i n layers of silica gel H d e v e l o p e d in toluene:ethyl a c e t a t e : e t h anol (4:1:1) at 4°C. In this solvent M P E m o v e d a h e a d of Pchlide. T h e M P E a n d Pchlide pools were eluted in m e t h a n o l : a c e t o n e (4:1). Chlorophyll a and b extraction and separation. Lightgrown c u c u m b e r leaves were e x t r a c t e d in 90% acetone. T h e e x t r a c t was c e n t r i f u g e d at 10,000 r p m at 4°C for 5 min. T o t h e s u p e r n a t a n t an equal v o l u m e of h e x a n e was a d d e d in a s e p a r a t i n g funnel. T h e t o p h e x a n e p h a s e was t a k e n a n d dried u n d e r N2 gas. T h e above p i g m e n t was c h r o m a t o g r a p h e d on t h i n layers of cellulose developed in p e t r o l e u m e t h e r (60-80):isopropanol (99:1, v/v). In this s o l v e n t Chl a m o v e d a h e a d of Chl b. Chl a a n d Chl b pools were eluted in diethyl e t h e r (13). Preparation of Chlide. T h e Chlide pool consists of Chlide a + Chlide b. It was p r e p a r e d f r o m p u r e s a m p l e s of Chl a a n d Chl b b y c h l o r o p h y l l a s e action (1). T h e y were m i x e d in a p r o p o r t i o n of 3:1, to mimic t h e Chlide a:Chlide b pool p r e s e n t in green plants. T h e i r concent r a t i o n s were d e t e r m i n e d b y s p e c t r o p h o t o m e t r y . Spectrophotometry. A b s o r p t i o n s p e c t r a were recorded on a S h i m a d z u UV-260 s p e c t r o p h o t o m e t e r . F o r the calculation o f concentrations of p u r e s a m p l e s of P r o t o IX, M P E , a n d Pchlide in ether, extinction coefficients of 1.5 X 105 M -1 c m -1 (404 nm), 1.82 X 104 M -1 c m -~ (589 n m ) , a n d 3.56 X 104 M -1 c m -~ (624 nm), respectively, w e r e used. Diethyl e t h e r was used as a solvent. T h e e t h e r was dried u n d e r N2 gas a n d the required a m o u n t of H E A R was a d d e d to redissolve the p i g m e n t s . H E A R was u s e d as solvent since P r o t o IX, M P E , a n d Pchlide are q u a n t i t a t e d s p e c t r o f l u o r o m e t r i c a l y in H E A R . T h e Chlide (a + b) pool was also dissolved in H E A R a n d p r e p a r e d f r o m extinction coefficients (in 80% acetone) of 7.69 X 104 M -~ cm -~ (663 n m ) for Chlide a a n d 4.45 X 104 M -1 c m -1 (645 n m ) for Chlide b.

Spectrofluorometry. F l u o r e s c e n c e s p e c t r a were recorded in ratio m o d e in a c o m p u t e r - d r i v e n S L M A M I N C O 8000C s p e c t r o f l u o r o m e t e r having a p h o t o n c o u n t i n g device a n d were c o r r e c t e d for p h o t o m u l t i p l i e r t u b e sensitivity. R h o d a m i n e B was u s e d in the reference c h a n n e l as a q u a n t u m counter. A t e t r a p h e n y l b u t a d i e n e block was u s e d to adjust t h e voltage in b o t h t h e c h a n nels (i.e., s a m p l e as well as reference channel) to 20,000 c o u n t s p e r second at excitation a n d e m i s s i o n wave-

SPECTROFLUOROMETRIC ASSAY OF PLANT PIGMENTS lengths of 348 and 422 nm, respectively. To reduce the noise level, the sample channel photomultiplier tube was cooled to - 2 0 ° C by a thermoelectric cooling device. T h e emission spectra were recorded at excitation a n d emission bandwidths of 4 nm. The emission spectra of pigments dissolved in H E A R were recorded from 580 to 700 nm. The d a t a were stored in a micro computer (IBM PS 30) diskettes. Chemicals. P r o t o p o r p h y r i n IX was obtained from P o r p h y r i n Products (Logan, UT); 2,2'-dipyridyl, ALA, silica gel H, and cellulose were obtained from Sigma Chemical Co. (St. Louis, MO).

k1

P r o t o IX(E440/7'638) P r o t o IX(E4o o F63~) '

Chlide(E44o F6as) k3 = Chlide(E400 F633) ' k5 -

Calculation of Net Fluorescence Emission Amplitudes of Proto I X and Pchlide in Mixtures of Proto IX, Pchlide, and Chlide In H E A R (293°K), Proto IX has emission m a x i m u m at 633 nm, when excited at 400 n m (E4oo F633)- Under identical conditions Pchlide a n d chlide, when excited at 440 nm, fluoresce at 638 n m (E440 F638) a n d 675 n m (E44o Fs75), respectively. As Chlide includes two components, i.e., Chlide a a n d Chlide b, their individual concentrations were not determined from the fluorescence emission spectra. T h e fluorescence due to Proto IX a n d Pchlide can be determined from their emission spectra and calculated by appropriate a d a p t a t i o n of Eqs. [1] a n d [2]. The equations for Proto IX and Pchlide are Proto IX(E40s Fs33) E4os F63~ - C1(E440 F638) - C2(E44o Fs75) Ca

c~

[1]

[21

C o n s t a n t s C1, C2, Ca a n d C'1, C'2, C'3 were calculated from the primary constants of kl, k2, k3, kt, ks, k6 a n d k'l, k'2,

k'~, k',, k;, k's: 1

Cl--k2

K,=l-k-k5 K4-k2,

K2.K4

~-'~3 kl ~, Ks-

'

K2 K2.K5 C2-Ka' C3=K1+--"-~3

k3 K2=l-k-~2, k 1 • k9 k2

K3=k6 -

Chlide(E400 F675) ks = Chlide(E400 F6a3) k'2 = P r o t o IX(E400 Fs33) P r o t o IX(E44 s F6~8)

Pchlide(Etoo F6~3) k'~ -- Pchlide(E440 F638) '

Pchlide(E440 Fs75) k'4 = Pchlide(E44o Fs3s)

Proto IX(E,4 o F675) P r o t o IX(E440 Fs3 s) '

Chlide(E44o Fs75) k'6 = Chlide(E4, ° Fs3s) "

T h e numerical values of the above c o n s t a n t s were calculated and utilized in solving Eqs. [1] a n d [2] for determination of fluorescence of Proto IX (Eq. [3]) a n d Pchlide (Eq. [4]), respectively: Proto IX(E4so F633) = 1.021(E4ss F633) -

0.4268(E44 o Fs38) - 0.0184(E4ao F675)

[3]

Pchlide(E44o F63s) = 1.03(E4as Fs38) - 0.0458(E44o Fs33) - 0.0358(Ea4o F675)-

[4]

Conversion of Proto I X and Pchlide Emission Amplitudes to Proto I X and Pchlide Concentrations

Pchlide(E4ts F63s) E . o Fs~s - C'l(E,so Fs3~) - C 2 ( E . s Fs75)

Pchlide(E440 F6a8) k2 = Pchlide(E4oo Fsa3)

P r o t o IX(E440 F675) k4 = P r o t o IX(E400 Fs33)

Pchlide(E44o F675) Pchlide(E40o F633) '

Chlide(Etoo F633) Chlide(E4to F638) '

RESULTS

127

k3k5 k2

ka.

Like C 1, C2, and Ca , the values of C'1, C~, a n d C'3 m a y be calculated as described above from the p r i m a r y constants defined as

E q u a t i o n s [3] a n d [4] allow the calculation of emission amplitudes of Proto IX a n d Pchlide in pure samples as well as in mixtures of these two tetrapyrroles in the presence of a third interfering compound, Chlide. Figure 1 shows the fluorescence emission spectra (E4o0 and Eta0) of pure samples of Proto IX, Pchlide, a n d Chlide and mixture of Proto:Pchlide:Chlide at a molar ratio of 1:1:2. Using Eqs. [3] a n d [4], emission amplitudes of Proto IX a n d Pchlide in mixtures of P r o t o IX, Pchlide, and Chlide pigments m a y be calculated a n d t h e n converted to concentrations by reference to a standard calibration curve. T h e latter is constructed (a) by either having various concentrations of pure P r o t o IX a n d Pchlide or mixing known proportions of Proto IX a n d Pchlide in the presence of Chlide, (b) by recording E4oo a n d Ea40 emission spectra on each sample, (c) by calculating P r o t o IX(E400 F63~) a n d Pchlide(E440 F6~8) deconvoluted emission amplitude for each mixture with the help of Eqs. [3] a n d [4], and finally (d) by plotting Proto IX or Pchlide c o n c e n t r a t i o n on the abscissa against calculated net deconvoluted amplitudes of Proto IX a n d Pchlide on the ordinate. T h e various mix-

128

H U K M A N I AND T R I P A T H Y 633

• t/3 I.-

TABLE 1

638

A

,,~

B

"0./* 3 3

-n

E:D >. ¢r' ,,¢: nl.-

0.357

M e a n P e r c e n t a g e E r r o r o f E q . [1] for P r o t o I X i n t h e M i x t u r e of P r o t o I X , P c h l i d e , a n d C h l i d e

I

D

I

<

I

rn

I

I

I

~

n1.1.1 t,9 Z I.I.J {,D ID I.,U Q::

Z

o -J

._J I.i..

4 3 650 WAVE L E N G T H , n r n

700

580 600

6~5

"~ 3~--~ L L t J , i 580 600 650 WAVELENGTH,nm

[

t 700

633

z ~ / / I

J

580 600

t

i

650

WAVELENGTH, nm

~

% error between a m o u n t of Proto IX added and calculated

5 30 60 70 80 90 95

5.39 29.86 58.03 65.50 73.38 89.64 100.64

7.80 -0.46 -3.28 -6.43 -8.27 -0.40 5.93

Mean % error ± SD

-0.73 ___5.5

t

700 580 600

650

Note. T h e deconvuluted fluorescence emission amplitude (E400 F633) was used to determine the concentration of Proto IX. The concentration of Chlide was 100 n m and the concentration of Pchlide was adjusted so t h a t in each experiment Proto IX + Pchlide was 100 riM. Other experimental details were as described under Materials and Methods.

and 0.0038 for Pchlide (r = 0.992). These slopes closely m a t c h e d t h o s e d e r i v e d f o r p u r e s a m p l e s , i.e., 0 . 0 0 3 7 f o r Proto IX and 0.0039 for Pchilde. The concentrations of Proto IX and Pchlide were calculated using the slopes of the calibration curves. The mean percentage error of E q . [3] i n d e t e r m i n i n g P r o t o I X i n a m i x t u r e o f P r o t o I X , P c h l i d e , a n d C h l i d e w a s - 0 . 7 3 +_ 5 . 5 % ( T a b l e 1) a n d t h a t f o r P c h l i d e i n t h e a b o v e m i x t u r e w a s - 0 . 5 7 5 +_ 5 . 5 8 9 % ( T a b l e 2). T h e L o w e s t l i m i t o f e s t i m a t i o n o f Proto IX, MPE, and Pchlide was 1 nM. At high concent r a t i o n s , i.e., a b o v e 1 5 0 n M , d u e t o q u e n c h i n g a n d p r o b a bly due to energy transfer between pigments, the quan-

l/ \M

u ~D Lfl nO 00z,3

Amount of Proto IX calculated (riM)

j

er

t,

Amount of Proto IX added (riM)

700

WAVE L E N G T H , n r n

l. T h e room-temperature (293°K) fluorescence emission spectra of various tetrapyrroles in H E A R excited at 400 n m (E40o , - ) and 440 n m (E44o, - - -). (A) Pure sample of Proto IX; (B) pure sample of Pehlide; (C) ehlides a + b; (D) mixture of Proto IX (50 nM), Pehlide (50 nM), and Chlides a + b (100 nM) (1:1:2). The fluoreseence speetra were corrected for photomultiplier tube sensitivity. They were recorded in ratio mode at excitation and emission slit widths of 4 n m as described under Materials and Methods. The numbers in the lower and upper left margins of each panel are the reeorded minimum and maximum values of fluoreseence intensities and refer to the eounts per second in the sample channel/counts per second in the reference channel. FIG.

ture of Proto IX and Pchlide were adjusted so that Proto IX + Pchlide concentration was 100 nM. The Chlide was added to the above mixture at a final concentration of 100 nM. The curve which related the concentration of Proto IX or Pchlide on the abscissa to the calculated deconvoluted emission amplitudes on the ordinate was a straight line that passed through the origin and it exhibited a slope of 0.0037 for Proto IX (r = 0.980)

TABLE 2 M e a n P e r c e n t a g e E r r o r o f Eq. [2] f o r P c h l i d e i n t h e M i x t u r e o f P r o t o IX, P c h l i d e , a n d C h l i d e Amount of Pchlide added (nM)

Amount of Pchlide calculated (nM)

% error between Pchlide added and calculated

10 20 3O 40 70 90 95

9.55 21.19 31.28 38.05 74.94 85.69 88.22

-4.50 5.95 4.26 -4.87 7.05 -4.79 -7.13

Mean % error ± SD

-0.575 ± 5.589

Note. The deeonvuluted fluorescence emission amplitude (E~o F638) was used to determine the concentration of Pchlide. T h e concentration of Chlide was 100 nM a n d the concentration of Proto IX was adjusted so t h a t in each experiment Pchlide + Proto IX was 100 nM. Other experimental details were as described under Materials and Methods.

SPECTROFLUOROMETRIC ASSAY OF PLANT PIGMENTS titative r e l a t i o n b e t w e e n p i g m e n t c o n c e n t r a t i o n a n d fluorescence yield d e v i a t e d f r o m linearity. T h e r e f o r e , this m e t h o d o l o g y c a n n o t be followed for c o n c e n t r a t e d s a m ples a n d t h e p i g m e n t s m u s t be diluted in H E A R b e f o r e q u a n t i f i c a t i o n by s p e c t r o f l u o r o m e t r y . T h e lowest limit of quantification of t h e a b o v e p i g m e n t s is 150-200 nM by s p e c t r o p h o t o m e t r y .

Sample Calculation of Amounts of Proto I X and Pchlide in Tetrapyrrole Mixture Containing Chlide A m i x t u r e of P r o t o IX, Pchlide, a n d Chlide h a v i n g final c o n c e n t r a t i o n s of 50, 50, a n d 100 nM, respectively, was p r e p a r e d in H E A R b y diluting t h e c o n c e n t r a t e d stock solutions quantified by s p e c t r o p h o t o m e t r y as described u n d e r M a t e r i a l s a n d M e t h o d s . T w o e m i s s i o n s p e c t r a were r e c o r d e d in H E A R at 293°K; one s p e c t r u m was r e c o r d e d at excitation m a x i m a of P r o t o I X at 400 n m a n d t h e o t h e r was r e c o r d e d at 440 n m for Pchlide. T h e n o n d e c o n v o l u t e d e m i s s i o n a m p l i t u d e s at 632 a n d 638 n m were m e a s u r e d . T h e d e c o n v o l u t e d n e t e m i s s i o n a m p l i t u d e s of P r o t o IX(E4oo F632) a n d Pchlide(E44o F638) were c a l c u l a t e d with help of Eqs. [3] a n d [4], respectively. T h e d e c o n v o l u t e d P r o t o IX(E4oo F6as) a m o u n t e d to 0.1785 fluorescence u n i t s a n d t h a t of Pchlide(E440 F63s) was 0.1906 fluorescence units. T h e a m o u n t of P r o t o I X a n d Pchlide was calculated f r o m t h e a b o v e fluorescence units of P r o t o I X a n d Pchlide a n d f r o m inverse of slopes of c a l i b r a t i o n curve, i.e., 1/0.0037 a n d 1/0.0038, as 0.1785/0.0037 = 48.25 n m ( P r o t o IX) a n d 0.1906/0.0038 = 50.16 n m (Pchlide). T h e s e closely matched the amounts measured spectrophotometrically.

Calculation of Net Fluorescence of MP(E) M g - p r o t o a n d M P E h a v e n e a r - i d e n t i c a l fluorescence spectral p r o p e r t i e s . In H E A R (at 293°K), M P ( E ) excited at 420 n m does n o t h a v e a s h a r p p e a k a n d it is a r o u n d 589-592 nm, a n d this fluorescence b a n d e x t e n d s f r o m 580 to 610 nm. T h e o t h e r c o m p o n e n t s , such as P r o t o I X a n d Pchlide, p r e s e n t in H E A R do n o t s u b s t a n tially fluoresce in t h e a b o v e w a v e l e n g t h range. T h e r e fore the fluorescence e m a n a t i n g f r o m t h e M P E was int e g r a t e d f r o m 580 to 610 n m . T h e i n t e g r a t i o n values of different c o n c e n t r a t i o n s of p u r e s a m p l e s of M P E were c o n v e r t e d to the c o n c e n t r a t i o n s b y r e f e r e n c e to a s t a n d a r d c a l i b r a t i o n curve. T h e l a t t e r was c o n s t r u c t e d (a) b y p r e p a r i n g various c o n c e n t r a t i o n s of M P E , (b) by recording one 2 9 3 ° K e m i s s i o n s p e c t r u m (E42o) on e a c h c o n c e n t r a t i o n , (c) b y calculating the a r e a u n d e r t h e fluorescence e m i s s i o n b a n d b e t w e e n 580 a n d 610 n m , a n d (d) by p l o t t i n g t h e c o n c e n t r a t i o n on t h e a b s c i s s a a g a i n s t

129

TABLE 3 Mean Percentage Error of Standard Curve which Was Constructed from Authentic Concentrations of the Pure Samples of MPE Amount of MPE added (nM)

Amount of MPE calculated (nM)

% error between amount of MPE added and calculated

10 20 30 40 50 60 70 80 90

11.33 21.66 31.89 41.69 47.63 62.34 70.12 78.33 87.59

13.30 8.30 6.3O 4.22 -4.74 3.90 0.17 -2.08 -2.67

Mean % error +_SD

2.96 +_5.5098

Note. The integration values from 580 to 610 nm (E420)were used to determine the concentration of MPE.

t h e c a l c u l a t e d a r e a on t h e ordinate. Such a p l o t yielded a s t r a i g h t curve t h a t p a s s e d t h r o u g h t h e origin a n d exhibited a slope of 0.73 a n d a coefficient of c o r r e l a t i o n o f 0.998. T h e m e a n p e r c e n t a g e e r r o r of this c a l i b r a t i o n c u r v e in d e t e r m i n i n g M P E was 2.96 + 5.5098% ( T a b l e 3). T h e c o n c e n t r a t i o n of M P E c a n be c a l c u l a t e d utilizing t h e slope d e s c r i b e d for P r o t o I X a n d Pchlide. DISCUSSION I n g r e e n p l a n t s , f r e q u e n t l y e n c o u n t e r e d fluorescing c o m p o n e n t s of t h e i n t e r m e d i a t e s of t h e Chl b i o s y n thetic p a t h w a y are P r o t o IX, M P E , Pchlide, a n d Chlide. T h e l a t t e r is u s u a l l y p r e s e n t in relatively larger q u a n t i ties a n d m a y be e s t i m a t e d b y s p e c t r o p h o t o m e t r y . As t h e s e p i g m e n t s h a v e o v e r l a p p i n g fluorescence s p e c t r a , it is essential to c o r r e c t for the s a m e . T h e p r e s e n t invest i g a t i o n d e m o n s t r a t e s t h e sensitivity a n d r e s o l u t i o n of r o o m - t e m p e r a t u r e fluorescence s p e c t r o s c o p y as a n a n a lytical tool for t h e q u a n t i t a t i v e e s t i m a t i o n of t e t r a p y r roles. It is o f t e n e x t r e m e l y difficult to s e p e r a t e m i n u t e q u a n t i t i e s of p i g m e n t s c h r o m a t o g r a p h i c a l l y , w h i c h s o m e t i m e s results in e i t h e r loss or d e s t r u c t i o n of t h e p i g m e n t s . T h e r e f o r e , t h e e l i m i n a t i o n of t h e n e e d for sepa r a t i o n a n d p u r i f i c a t i o n of v a r i o u s t e t r a p y r r o l e s p r i o r to q u a n t i t a t i v e a n a l y s i s of p i g m e n t s is a u n i q u e f e a t u r e of t h e a b o v e p r o c e d u r e . As t h e c o n c e n t r a t i o n s of P r o t o IX, M P E , a n d Pchlide in 80% a c e t o n e e x t r a c t s o f chlorop l a s t s are m i n u t e , i.e., only a few n a n o m o l e s p e r liter, t h e y c a n n o t b e q u a n t i f i e d b y s p e c t r o p h o t o m e t r y . As this s p e c t r o f l u o r o m e t r i c t e c h n i q u e c a n e s t i m a t e as low as 1 nM c o n c e n t r a t i o n of p i g m e n t s f r o m t h e i r m i x t u r e s , it offers significant a d v a n t a g e o v e r o t h e r available m e t h o d s . C o n v e n i e n c e , rapidity, a n d a c c u r a t e q u a n t i t a t i o n of m i n u t e (1 nM) c o n c e n t r a t i o n s of p i g m e n t s a n d

130

HUKMANI AND TRIPATHY

e l i m i n a t i o n of a n a l y t i c a l u n c e r t a i n t i e s d u e t o r e c o v e r y losses caused by c h r o m a t o g r a p h y are the u n i q u e a d v a n t a g e s of t h e s p e c t r o f l u o r o m e t r i c m e t h o d of a n a l y s i s of pigments.

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