The P700-chlorophyll a-protein

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

165, 388-397

(1974)

The P700-Chlorophyll Isolation JUDITH

and Some

A. SHIOZAWA,

Department

o/Biology

Characteristics RANDALL

and Molecular

Biology

a-Protein of the Complex

S. ALBERTE, Institute,

Received

University June

AND of California,

in Higher J. PHILIP Los

Angeles,

Plants THORNBER California

L?M).L?4

17, 1974

A P700chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100.dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662,669, 677, and 686 nm, a chlorophyll/P700 ratio of 40-45/l, and contains only chlorophyll a and p-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b, are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.

The major chlorophyll-protein complex in sodium dodecylsulfate (SDS) extracts of blue-green algae is the P700-chlorophyll a-protein complex (1, 2). This algal complex has been isolated in a homogeneous form and in sufficient quantities for studies to be made of its composition (1) and function (2). The SDS-soluble complex of 110,000 daltons is composed only of chlorophyll (Y, p-carotene, and protein; this chlorophyll-protein has a red wavelength maximum at 677 nm, a chlorophyll/P700 ratio of 40-4511 (using a differential extinction coefficient for I’700 of 64 mM-’ cm-’ at 697 nm (3)), and a chlorophyll/protein ratio of 14 moles of chlorophyll a per 110,000 g of protein. Higher plants contain a chlorophyll a-protein termed “Complex I” (4). Thornber (1) postulated on the basis of similar sizes and amino acid and pigment compositions that Complex I and the blue-green algal P700-chlorophyll a-

protein were homologous; however, Complex I could not be isolated from any eucaryotic organism with the same spectral composition nor with the same enrichment of P700 obtained for the P700-chlorophyll a-protein of blue-green algae (cf. Refs. 5 and 6). The aim of this paper was to determine whether it was possible to isolate a P700chlorophyll u-protein from higher plant chloroplasts with characteristics homologous to the blue-green algal complex. If this aim was accomplished, it would then be possible to study the relationship between the previously described higher plant Complex I derived from SDS-dissolved chloroplast lamellae and the newly isolated P700-chlorophyll a-protein. MATERIALS Isolation Nicotiana 388

Copyright All

rights

0

1974 by of reproduction

Academic in any

Press, Inc. form

resewed.

AND

METHODS

of chloroplast lamellae. Young leaves of tabacum, corn (Zea mays), jack bean

P700-CHLOROPHYLL (Canaualia ensiformis (L.) (DC.), bush bean (Phaseolus uulgaris), pea (Pisum satiua), and barley (Hordeurn uulgare) were obtained from greenhousegrown plants, and pine (Pinus radiata) needles were obtained from mature trees. Leaves were homogenized in an isolation medium consisting of 0.5 M sucrose, 0.1 M NaCl, 50 mM Tris-HCl, 20 mM sodium ascorbate (pH 8.0). The brei was filtered through four layers of Miracloth. Cellular debris was removed by centrifugation at 3000g for 1 min. Chloroplasts were pelleted by centrifugation at 20,OOOg for 10 min and then resuspended in 0.1 M NaCl, 50 mM Tris-HCl, 20 mM sodium ascorbate, 1 mM EDTA (pH 8.0). The suspension was spun at 30,OOOg for 20 min. This procedure was repeated. The resulting pellet was either used immediately or transferred to a minimal volume of 50 mM Tris-HCl containing 20 mM sodium ascorbate (pH 8.0) and stored below 0°C. Isolation of the P700-chlorophyll a-protein. The procedure is summarized in the flow diagram (Fig. 1). Chloroplast lamellae were solubilized in 1% Triton X-100, 50 mM Tris-HCl, 20 mM ascorbate (pH 7.4) using a Kontes glass homogenizer; the detergent to chlorophyll ratio was 75/l (w/w). The extract was stirred for IO-15 min at 4°C and then centrifuged at

4) 0.2 M scdium phoephate (pli 7.0) e1ution until e1uate is colorle.%5

389

a-PROTEIN

27,OOOg for 10 min. The pellet which contained mostly starch was discarded. The supernatant (about 150 ml) was loaded on a hydroxylapatite (7) column (3.0 x 4.0 cm) which was previously equilibrated with 10 mM sodium phosphate (pH 7.0). About two-thirds of the total chlorophyll was not adsorbed on the hydroxylapatite and was washed from the column with 10 mM sodium phosphate until the eluate was colorless. At this point there was a tightly bound pigmented band at the top of the column. Next, the column was washed with 1% Triton in 50 mM Tris plus 20 mM ascorbate (pH 7.4); the wash volume was about 1.5 times the volume of the starting extract. The Triton wash released about 20% of the bound chlorophyll. The column was then washed with 10 mM sodium phosphate until a colorless eluate was obtained. The P700-chlorophyll a-protein was eluted from the column with 0.2 M sodium phosphate (pH 7.0). Gel electrophoresis. Methods for SDS-polyacrylamide gel electrophoresis were the same as those described (8) with the exception that the buffer system used here consisted of 0.1% SDS in 50 mM TrisHCl plus 2 mM MgCI, (pH 8.0). Spectrophotometry. All spectrophotometric measurements were made on an Aminco DW-2 dual-

,

FIG. 1. Flow diagram for the isolation of the P700-containing eluate) from Triton X-lOO-solubilized chloroplast lamellae.

fraction

(0.2 M sodium

phosphate

390

SHIOZAWA,

ALBERTE

wavelength, double-beam spectrophotometer. Chlorophyll content and chlorophyll a/b ratios were determined in 80% acetone using Arnon’s (9) equations. Chlorophyll concentration was estimated for P700 measurements using an extinction coefficient of 60 mM- I cm- ’ at the red peak (cf. Ref. 1). The millimolar absorptivity values of the P700chlorophyll a-protein were determined using methods previously described (1). The concentration of P700 was determined from the reversible light-induced absorbance change at 697 nm, using an isobestic point of 725 nm as a reference. An extinction coefficient of 64 rnM-’ cm-’ (3) was used. The actinic light was filtered through a blue Corning filter (no. 5433); a filter (Corning no. 2404) which blocked the actinic light was placed between the sample and the phototube. Rates of light oxidation and dark recovery of P700 were facilitated by addition of sodium ascorbate, dichlorophenol-indophenol, and methyl viologen (cf. Ref. 2). Liquid-nitrogen spectra of the isolated chlorophyllprotein were used for computer-assisted curve-fitting analysis using Gaussian components after the procedures of French et al. (10). The relative proportion, wavelength position, and half-band width of each spectral component were obtained from the computer program. Cytochrome f was detected from potassium ferricyanide vs sodium ascorbate difference spectra; its concentration was determined by using 17.7 mM-’ cm-’ (11) as the differential extinction coefficient at 554.5 nm. Cytochrome b, was detected from sodium ascorbate vs sodium dithionite spectra; an assumed differential extinction coefficient of 20 mM-’ cm-’ (cf. Ref. 11) was used at 563 nm. Pigment analysis. Carotenoids in the isolated chlorophyll-protein were quantitated by the procedures of Davies (12). The concentrated pigment extract was applied to Silica Gel IB sheets, developed by ascending chromatography in petroleum ether:acetone (3:l. v/v), and R, values obtained. The bands were removed from the sheets and eluted with absolute ethanol. The separated carotenoids and chlorophylls were then identified by their R, values and absorption characteristics. Ultracentrifugation. Sedimentation-velocity experiments on the isolated material were performed in a Beckman Spinco Model E analytical ultracentrifuge. RESULTS

Gel electrophoresis of SDS chloroplast extracts. It has been shown (4, 13) that photosynthetic membranes of higher plants dissociated with anionic detergents yield three green bands upon electrophoresis in SDS-polyacrylamide gels (Fig. 2). The

AND

THORNBER *TOP

-

COMPLEX

I

-CHLOROPHYLL -FREE

a/b-PROTEIN

PIGMENT

FIG. 2. Polyacrylamide gel electrophoresis pattern of SDS extracts of tobacco lamellae. This unstained gel shows the position of Complex I, the light-harvesting chlorophyll a/b-protein, and of the free pigment zone. Direction of electrophoresis was from top to bottom, i.e., toward the anode.

green zone of lowest electrophoretic mobility in the system is Complex I, a chlorophyll u-protein, also referred to as CPI or the photosystem I chlorophyll-protein. The zone of intermediate mobility is termed Complex II, CPII, or the photosystem II chlorophyll-protein; recently this chlorophyll-protein has been renamed the major light-harvesting chlorophyll a/b-protein (8). The zone of fastest mobility is free pigment complexed with detergent. The percentage of chlorophyll associated with each zone is shown in Table I. The absorption spectra of the bands have been reported previously (8). Complex I has an absorption spectrum (cf. Fig. 4) as well as an electrophoretic mobility essentially identical to those of the P7OOchlorophyll u-protein of blue-green algae. A major difference between these two spectra is in the location of the red wavelength maximum-677 nm in the algal moiety and 671 nm in the higher plant Complex I. Isolation of the P700-chlorophyll a-protein of higher plants. The presence of the chlorophyll-protein was monitored by absorption spectrophotometry. The criteria of purity were that the red wavelength maximum of the desired material should be at 677 nm, a shoulder should be present in the spectrum at 500 nm, but there should be an absence of any peak or shoulder at 470 nm (the Soret peak of chlorophyll b),

P700-CHLOROPHYLL

and the chlorophyll/P700 ratio in the purified material should be 40-4511. Alternative detergents to SDS were tested for their suitability to provide material that meets these criteria. Triton X100, which is used extensively by Vernon and his colleagues (14) and by Loach and his coworkers (15) to dissociate photosynthetic membranes and to provide a separation of photosystems I and II, was tried initially. Triton-dissociated chloroplast membranes were applied onto hydroxylapatite, a chromatographic medium which has proved successful for the resolution of several other detergent-soluble pigment-proteins of plants and bacteria (5). The component for which we were searching was adsorbed to the hydroxylapatite whereas most of the other pigmented material was not. The adsorbed P700-containing material was partially removed from the column with 0.2 M sodium phosphate (pH 7.0); however, a small amount of chlorophyll b was present in the eluate. Consequently, the isolation procedure was varied in an attempt to eliminate chlorophyll b from the eluate: (a) the Triton/chlorophyll TABLE

I

PERCENTAGES OF CHLOROPHYLL PRESENT IN EACH ZONE FOLLOWING POLYACRYLAMIDE GEL ELECTROPHORESIS OF SDS EXTRA(TPS OF PHOTOSYNTHETIC MEMBRANES OF JACK BEAN AND TOBACCO LEAVES (CF. FIG. 2)

Complex I Light-harvesting chlorophyll a/b-protein Free pigment

Jack bean

Tobacco

13 zt 1% (16) 50 i 3% (16)

12 + 1 (16) 52 i 3 (16)

37 zt 3% (16)

36 i 3(16)

TABLE RECOVERY

Triton extract 10 mM sodium phosphate 1% Triton eluate 0.2 M sodium phosphate o Molar

ratio.

eluate eluate

391

a-PROTEIN

ratio used to dissociate the membranes was varied between 6 and 300/l; (5) the effect of incubation time in detergent at different pH values was tested; (c) the extent of washing the complex with detergent while adsorbed to the hydroxylapatite was studied; (d) and other detergents (BRIJ 35 and 36T, Lubrols PX and WX, cetyl trimethyl ammonium bromide, sodium deoxycholate, and Ammonyx LO) were tested. It was concluded that the 0.2 M sodium phosphate eluate from hydroxylapatite chromatography of Triton X-lOO-dissociated membranes (Triton/chlorophyll = 75/l (w/w)) gave the highest yields of a component possessing the spectral criteria. The details of the isolation of the desired component are summarized in Fig. 1. Yield of P700-containing material. Recoveries from all chromatographic steps in a typical experiment are given in Table II. The recoveries of P700 and chlorophyll in the 0.2 M sodium phosphate eluate were 45 and 5%, respectively. On some occasions, slightly more chlorophyll was eluted by 1% Triton and more P700 was obtained in the 10 IIIM sodium phosphate washes; the yield of P700 in the purified material can vary between 30 and 550/o, but the chlorophyll/P700 ratio (40-45/l) remains constant in every preparation. Some green material, presumably containing some P700, remains tightly bound to the column after the chromatography. If the recovery of P700 in the 0.2 M sodium phosphate eluate had been lOO%, then the amount of associated chlorophyll would agree very well with the proportion (12%) of the total chlorophyll associated with Complex I determined from gel scans (Table I). Absorption spectrum of the isolated II

OF P~O@CHLOROPHYLL

(I-PROTEIN

Chlorophyll (%)

Chl;,;phyll

100 66 17 5

2.9 2.9 1.6 >7

u

Chlorophyll/ P700” 360/l 900/l 4;)/1

P700 (%I 100 27 0 45

392

SHIOZAWA.

ALBERTE

material. The room-temperature absorption spectra of the purified material and of the P700-chlorophyll a-protein of bluegreen algae are shown in Fig. 3. The millimolar absorptivity values for the higher plant and blue-green algal complexes are compared in Table III. The two preparations show essentially identical spectral characteristics. Both have a red wavelength maximum at 677 nm with the same extinction coefficient, both show a shoulder at 500 nm due to the presence of P-carotene, and both appear to be free of chlorophyll b. Slight differences between the two complexes can be detected from the room-temperature measurements: the absorbance at 420 nm is a peak in the higher plant material whereas in the algal complex it is only a shoulder; this difference is also reflected in the millimolar absorptivity values (Table III). The presence of the Soret band of the cytochromes in the spectrum probably accounts for the variation in the 420-nm absorbance since the higher plant complex contains cytochromes whereas the algal complex does not. At present, we do not know the reason for the large difference in absorptivity values at 340 nm. Liquid-nitrogen-temperature spectra (Figs. 3 and 4) reveal that the two complexes differ in their content of the longer wavelength spectral forms of chlorophyll

AND

THORNBER

a-a definite shoulder at 705 nm is present in the algal complex but is missing from the higher plant material. These longerwavelength spectral forms are known to be susceptible to detergent treatment and to vary in their stability in different organisms (16, 17). The major spectral forms of chlorophyll a in the purified material were delineated by computer-assisted curve fitting to be C, 662, 669, 677, and 686 nm. These are the four major chlorophyll a spectral forms thought to be present in all plants (10). The spectrum in Fig. 4 shows that C, 686 is particularly enriched in the purified material. Preliminary analysis of the same material by fourth-derivative spectrophotometry at liquid-nitrogen temperature (cf. Ref. 18) also showed the presence of four peaks at 662, 666, 677, and 684 nm. TABLE

III

MILLIMOLAR AESORPTIVITY VALUES~ OF P700-CHLOROPHYLL U-PROTEIN BASED ON CHLOROPHYLL a Wavelength

342 c (mM-’

420

437

629

677

56

85

88

15

59

35

75

78

15

60

Cm-‘)

Tobacco Blue-green a Limit

(nm)

alga of error

estimated

as 13%.

FIG. 3. Room-temperature absorption spectrum of the P700xontaining fraction of tobacco chloroplast lamellae. Inset shows the room temperature and 77°K spectra of the P700-chlorophyll a-protein isolated from SDS-solubilized P. luridurn photosynthetic membranes (cf. Ref. 2).

P7OOCHLOROPHYLL

151

153 149 145 WAVENUMBER (cm 1 x IO 21

141

FIG. 4. Computer-assisted curve analysis of the 77°K absorption spectrum of the tobacco 0.2 M sodium phosphate eluate. Absorption maxima of the component curves are indicated and the error of fit for each point is shown on the scale below (provided courtesy of Dr. J. S. Brown, Carnegie Institution of Washington, Stanfrod, CA).

Addition of SDS to the purified material (Fig. 5) shifted the red wavelength maximum from 677 nm to shorter wavelengths. After 30-min incubation in SDS, the red maximum shifts to the lower wavelength maximum observed previously for Complex I; thereafter, pheophytinization begins (Fig. 5). Loss of magnesium from the chlorophyll molecules is seen in the relative increase in absorbance at 420 nm compared to that at 437 nm (Fig. 5) and in the appearance of the absorbance at 540 nm. Preliminary analysis by fourth-derivative spectrophotometry of the isolated material treated with SDS confirms that indeed the longer wavelength-absorbing forms in the complex are lost. Addition of SDS to the algal P700chlorophyll u-protein, however, does not change its spectral characteristics. Photochemical activity of the isolated material. P700 occurs in the isolated material (Table II). The chlorophyll/P700 ratio is invariably found to be 40-45/l representing a 7- to lo-fold enrichment of P700 in

a-PROTEIN

393

the isolated material. This ratio has been observed to be the same in material isolated from either jack bean, tobacco, barley, pea, maize, or pine leaves and it is identical to that in the P700-chlorophyll u-protein of the blue-green algae. Comparison of the light-induced difference spectrum of the isolated material with that obtained previously (2) for the bluegreen algal complex shows no significant differences. The kinetics of the light-induced absorbance changes at 697 nm in the higher plant material are compared with those of the blue-green algal complex prepared by SDS treatment in Fig. 6. The full extent of the change is observed in the higher plant maberial in the absence of any additives; however, the algal complex requires the addition of a source of electrons (sodium ascorbate) as well as an electron acceptor (methyl viologen) or of phenaxine methosulfate (cf. Ref. 2) to see the full extent of the bleaching. Addition of SDS to the material isolated from higher plants decreases the measurable P700. On prolonged exposure of the complex to solutions containing SDS/ chlorophyll ratios greater than 100/l (mole/mole) most of the detectable P700 is lost. Composition of the Isolated Material. (a) Pigments. The chlorophyll a/b ratio of the isolated material was greater than 7/l, a value at which the reliability of the equations used for the determination of the concentration of chlorophyll b is poor. Thin-layer chromatography of the pigments in the material showed no chlorophyll b was present in most preparations. Beta-carotene occurred in all preparations in a ratio of 1 mole of P-carotene per 35 moles of chlorophyll a. Complex I of higher plants and the blue-green algal P700chlorophyll u-protein contain 1 mole of p-carotene per 30 moles of chlorophyll a (1). (b) Cytochromes. Cytochromes f and b, were observed in chemically oxidized vs reduced difference spectra of the isolated material (P700:cyt f:cyt b, = 1:0.9&0.2: 1.8hO.3); no cytochromes are present in the blue-green algal material.

394

SHIOZAWA,

ALBERTE

AND

THORNBER

10

350

450

550 WAVELENGTH lnmj

650

750

FIG. 5. Changes in the absorption spectrum of the P700containing fraction from tobacco the presence of 500 moles of SDS per mole of chlorophyll. Spectra shown are after 0 min (---), min (-----), and 155 min (- .- .-) of incubation at room temperature in the darks.

P LURIOIJM

‘T-f--

N TABACUM

c

no addltlons

off

t

off

F +ascorbate

v

4

v

+methyl

c

vlalogen

FIG. 6. Comparison of the kinetics of the lightinduced absorbance changes at 697 nm in P700chlorophyll a-protein complexes of P. luridurn and tobacco with and without additions of electron donors and acceptors.

in 37

Gel electrophoresis. Electrophoresis of the 0.2 M sodium phosphate eluate on SDS-polyacrylamide gels shows two pigmented zones after about 40 min. The pigmented band of slower migration shows the same electrophoretic mobility and characteristic bluish-green color as the algal P700-chlorophyll u-protein. The other pigmented zone consists of free pigment released from the more slowly moving chlorophyll-protein moiety by the action of the detergent (cf. Fig. 2). Absorption spectra of the bluish-green band in situ reveal that this component has the same spectral characteristics as the previously described higher plant Complex I and the same as those of the blue-green algal component with the exception of the red wavelength peak position. Upon staining the gels, the upper band binds the protein dye while no stain is observed in the free pigment region. In many instances three or four other protein zones of smaller size than the pigmentprotein complex are delineated. Presently it is felt that these other proteins are cytochromes and/or subunits of the P700chlorophyll u-protein complex. Further studies are in progress to identify the other protein constituents. Ultracentrifugation. Material obtained

P700-CHLOROPHYLL a-PROTEIN directly from the hydroxylapatite column gave the Schlieren pattern shown in Fig. 7. Only one boundary of sedimentation coefficient, l3S, was observed in the sample. The pigments in the solution sedimented with this boundary. The blue-green algal complex in 0.2% SDS-containing buffers has a sedimentation coefficient of 9.1s. DISCUSSION One of the aims of this study was to isolate material from higher plant chloroplasts exhibiting spectral characteristics that were precisely analogous to those of the P700-chlorophyll u-protein of bluegreen algae. This was accomplished: hydroxylapatite chromatography of Triton X-loo-dissociated chloroplast membranes has yielded material that shows a red wavelength maximum at, 677 nm (Fig. 3), contains P700 in a ratio of one P700 per 40-45 chlorophyll molecules (Table III), and exhibits a P700 difference spectrum identical to that of the algal complex. These results plus other data on the chemical composition and physical properties of the P700-chlorophyll a-protein preparations from blue-green algae and higher plants permit the conclusion to be made that, whereas the algal component is a chlorophyll-protein composed of only chlorophyll a, P-carotene, and protein, the higher plant material is a multienzyme

FIG. 7. Schlieren pattern obtained upon ultracentrif’ugation of the 0.2 M sodium phosphate (pH 7.0) eluate from hydroxylapatite chromatography of’ Triton X-100.solubilized tobacco lamellae. The photograph was taken 15 min after reaching a speed of 54.770 rpm.

395

complex in which a P700-chlorophyll aprotein, homologous to the algal complex, is the major component and cytochromes f and 6, and possibly other substances are minor components. This difference between the two preparations explains the variation in their absorption spectra in the 300-450-nm region (Fig. 3 and Table III) as well as the presence of some additional protein bands upon gel electrophoresis of the higher plant complex. The presence of additional components in the P700-chlorophyll u-protein complex of higher plants can also explain its higher S value and may explain the difference in the kinetics of P700 photooxidation (Fig. 6). Further comparative analysis of the electron transfer reactions of the procaryotic and eucaryotic complexes may prove very important to an understanding of electron transport associated with photosystem I. Although SDS is suitable for the isolation of the blue-green alga P700-chlorophyll aprotein (l), it is clear from the data presented here that SDS destroys the spectral properties of the higher plant complex (Fig. 5). Thus, these observations explain why the P700-chlorophyll u-protein with the desired spectrum and P700 content cannot be isolated from SDS-treated chloroplasts (cf. Ref. 5). It is hypothesized that the lower cysteine concentration in the higher plant material (1) results in the absence of a crucial disulfide bridge which prevents SDS from unraveling the polypeptide chain(s) in the algal complex. Several characteristics-the action of SDS on the higher plant complex (Fig. 5), the presence of Complex I upon electrophoresis of the Triton-isolated material, and the agreement in chlorophyll yield between the isolated P700-chlorophyll uprotein (assuming 100% yield, see Results) and the amount of chlorophyll associated with Complex I from gels (Table I)-provide persuasive evidence that the previously described Complex I represents a SDS-altered form of the P700-chlorophyll u-protein from higher plants. Furthermore, if Complex I represents a cytochrome-lacking and spectrally altered form of the P700chlorophyll u-protein, then it is easy to rationalize the almost identical amino acid

396

SHIOZAWA,

ALBERTE

composition of Complex I and the bluegreen algal chlorophyll-protein (1). The close association of P-carotene with the P700-chlorophyll u-protein is apparent from this and previous work. The carotene almost certainly accounts for the shoulder at 500 nm in the absorption spectrum (Fig. 3). Its role in the complex is not understood, but it may be significant that P700 and B-carotene occur in nearly the same proportion in the complex. That this carotene is not essential for photochemical activity can be deduced from the work of Vernon and coworkers (14, 19, 20) whose HP700 fractions do not contain p-carotene. Because of their similar composition and spectral properties, HP700 and our higher plant fraction must be different preparations of the same entity. Both fractions are more enriched in P700 than other photosystern I preparations (15, 21-23). Our method, however, is more rapid than Vernon’s (19, 20) for the isolation of material of equivalent P700 enrichment. Furthermore, our preparation, unlike the HP700 fractions, lacks chlorophyll b and shows more rapid light-induced absorbance changes for the oxidation and reduction of P700 (Fig. 6). Thus, our procedure provides the heart of photosystem I with likely no alteration from its state in uiuo. Further substantiation of the native state of our complex is the presence of the same four spectral forms of chlorophyll (Fig. 4) in this isolated complex as found in intact chloroplast membranes (cf. Refs 10, 18). Clearly, if the in uiuo spectral forms are present, then it is reasonable to expect that the integrity of the photosystem I reaction center complex has been maintained through the isolation procedure. This research has demonstrated as conclusively as possible at the present that the P700-chlorophyll u-protein is homologous in higher plants and blue-green algae. In addition, concurrent research (24) has found that all other classes of algae examined also contain a P700-chlorophyll aprotein that behaves upon treatment with SDS like the higher plant fraction and not like the blue-green algal complex. Thus, it appears very likely that the heart of photosystem I is organized and possibly syn-

AND

THORNBER

thesized (25) in the same manner chlorophyll a-containing organisms.

in all

ACKNOWLEDGMENTS The research was supported by NSF Grant GB 31207; support for J.A.S. was provided by NDEA Title IV fellowship. The authors thank Dr. J. S. Brown for performing the curve analysis (Fig. 5), Prof. W. L. Butler and Dr. M. Kitajima for providing the fourth-derivative spectra, Dr. S. D. Kung for the ultracentrifugation, and Mrs. M. M. Frick for skilled technical assistance. REFERENCES

5. 6. 7.

8. 9. 10. 11.

12.

13. 14.

15. 16. 17. 18. 19.

THORNBER, J. P. (1969) Biochim. Biophys. Acta 172, 230-241. DIETRICH, W. E., JR., AND THORNBER, J. P. (1971) Biochim. Biophys. Acta 245, 482-493. HIYAMA, T., AND KE, B. (1972) Biochim. Biophys. Acta 267, 160-171. THORNBER, J. P., GREGORY, R. P. F., SMITH, C. A., AND BAILEY, J. L. (1967) Biochemistry 6, 391-396. THORNBER, J. P., AND OLSON, J. M. (1971) Photothem. Photobiol. 14, 329-341. KUNG, S. D., AND THORNBER, J. P. (1971) Biochim. Biophys. Acta 253, 285-289. SIEGELMAN, H. W., WIECZOREK, G. A., AND TURNER, B. C. (1965) Anal. Biochem. 13, 402-404. THORNBER, J. P., AND HIGHKIN, H. R. (1974) Eur. J. Biochem. 41, 109-116. ARNON, D. I. (1949) Plant Physiol. 24, l-15. FRENCH, C. S., BROWN, J. S., ANDLAWRENCE, M. C. (1972) Plant Physiol. 49, 421-429. BENDALL, D. S., DAVENPORT, H. E., AND HILL, R. (1971) in Methods in Enzymology (San Pietro, A., ed.), Vol. 23 pt. A, pp. 327-344, Academic Press, New York. DAVIES, B. H. (1965) in Chemistry and Biochemistry of Plant Pigments (Goodwin, T. W., ed.), p. 489, Academic Press, London. OGAWA, T., OBATA, F., AND SHIBATA, K. (1966) Biochim. Biophys. Acta 111, 223-234. VERNON, L. P., SHAW, E. R., OGAWA, T., AND RAVEED, D. (1971) Photo&em. Photobiol. 14, 343-357. LOACH, P. A., SEKURA, D. L., HADSELL, R. M., AND STEMER, A. (1970) Biochemistry 9, 724-733. BROWN, J. S., AND FRENCH, C. S. (1961) Biophys. J. 1, 539-550. GOEDHEER, J. C. (1973) Biochim. Biophys. Acta 314, 191-201. BUTLER, W. L., AND HOPKINS, D. W. (1970) Photo&em. Photobiol. 12, 439-450. YAMAMOTO, H. Y., AND VERNON, L. P. (1969) Biochemistry 8, 4131-4137.

P700-CHLOROPHYLL 20. VERNON, L. P., YAMAMOTO, H. Y., AND OGAWA, T. (1969) J’roc. Nut. Acad. Sci. USA 63, 911-917. 21. BOARDMAN, N. K. (1970) Annu. Reu. Plant Physiol. 21, 115-140. 22. MICHEL, J. M., AND MICHEL-WOLWERTZ, M. R. (1968) Carnegie Inst. Wash. Yearb. 67,50%514. 23. WESSELS, J. S. C. (1966) Biochim. Biophys. Acta

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126, 581-583. 24. BROWN, J. S., ALBERTE, R. S., THORNBER, J. P., AND FRENCH, C. S. (1974) Carnegie ht. Wash. Year-b. 73, (in press). 25. ALBERTE, R. S., THORNBER, J. P., AND NAYLOR, A. W. (1973) Boc. Nat. Acad. Sci. USA 70, 134-137.