Photochemrriry and Phorohrology. Vol. 29. pp 41Y 411. 0 pcrg:.,mon Prc*\ L ld . 1979. Prinicd in Grwt Brikiin

TECHNICAL NOTE

A SIMPLE MODIFICATION OF THE SPECTROPHOTOMETER FOR RAPID PHYTOCHROME

ASSAY

J I N JUNG and PILL-SOON SONG Department of Chemistry, Texas Tech University. Lubbock. TX 79409. U S A .

(Recrirc4 I t April 1978: ticcryfed 26 June 1978)

Abstract-A simple. low cost modification of a conventional single wavelength spectrophotometer (e.g. Cary 118C) enables one to quantitate phytochrome in crude extracts and purified phytochrome prep- arations without the use of a dual-wavelength ratio spectrophotometer.

INTRODUCTION

Since the dual-wavelength ratio spectrophotometer was described by Butler ef a/ . (1964) for phytochrome assay, expensive commercial as well as custom-built ratio-spectrophotometers of various types with essen- tially identical principles and designs have been employed in several laboratories (Spruit. 1970: Kidd and Pratt, 1973: Pratt and Marme, 1976: see review, Kendrick and Spruit, 1977). Although conventional, single-wavelength spectrophotometers have been used frequently by Butler et a/ . (1959) and others because of their ready access, they suffer from the major disad- vantage that they display the total absorbance to which phytochrome contributes very little, unless phytochrome is at least partially purified.

We describe here a simple modification of a con- ventional, single-wavelength spectrophotometer (Cary 118C) for rapid phytochrome assay. The modification is based on the fact that the phototransformation of phytochrome is monitored uniquely, without severe interference due to other absorbing material, by double-difference specta. Figure 1 will serve t o illus- trate the double difference spectrum of partially puri- fied phytochrome (A660/A278 = 0.15).

METHODS

Crude extracts containing phytochrome are preirra- diated with red light so as to convert P, to PI, and to saturate photoconversion of other photoirreversible pig- ments. After the red light treatment, the following pro- cedure is carried out.

Difference spectra are recorded after irradiating the sample and reference appropriately using the device de- scribed below (Fig. 2) and appropriate filters (Oriel C572-6600 1660 nm] and Ealing 26-4457 [720 nm]). Differ- ence spectrum 1 (DS-I) is recorded after irradiating the sample with far red light and the reference with red light. Difference spectrum 2 (DS-2) is then recorded after switch- ing the filters so that the sample is irradiated with red light while the reference is irradiated with far red light.

DS-I and DS-2 make up the "double" difference spectrum and the magnitude of the difference between them ( A A ) is proportional to phytochrome concentration. The rela- tionship between the double difference spectrum and phytochrome concentration can be calculated from pub- lished extinction coefficients for phytochrome (Tobin and Briggs, 1973). In this way. the following relationship can be established: (Phytochrome),,,,,, = (1.33/~!&~)AA~~,, = (1 .07/~!&~)6A,,,

( 1 ) where ~2~~ is the extinction coefficient of P, at 667nm. The absorbance of irradiated phytochrome may depend upon the actinic wavelength (Butler rf ul.. 1964).

Figure 3 illustrates the double difference spectral charac- teristics of the early stage of phytochrome isolation and

I . I A

1 DS-I A

006 1

Wavelength, nrn

Figure 1. The absorption (lower panel) and double diner- ence (top panel) spectra of phytochrome in 0.1 M sodium

phosphate buffer (pH 7.8).

419

420 Technical note

Lamp

I

Fil

Cory IISC Sample Monitoring beams compartment

Figure 2. The inside front view of the double difference spectra attachment (top) with black painted housing hold- ing filters and light source. Two cuvettes positioned inside the Cary 118C spectrophotometer cell compartment (lower box. dotted line) are also shown. Dimension of the attach-

ment is 20.5 x 10.5 x 10.5cm.

purification (Pratt and Coleman. 1971: Tobin and Briggs. 1973: see reviews by Furuya, 1976 and Pratt. 1978) and clearly demonstrates the resolving capacity of the double difference spectrum. It should be noted that. at this stage (i.e. first tissue extracts in the isolation procedure). the total absorption spectrum does not reveal any hint of phyto- chrome due to its extremely low concentration masked by interfering pigments and light scattering (Fig. 3). From the double difference spectra of a highly enriched oat phyto- chrome preparation from the Brushite column (spectra not shown). the phytochrome level was found to be 0.04 units (cf. Pratt. 1978) or 0.055 mgjm/ using the specific absorp- tion coefficient of ca. 0.6m//mg/cm at 667nm and Eq. I .

From these observations, it can be concluded that the double difference spectra device used (ride inf i l l ) makes a conventional, single wavelength spectrophotometer a con- venient instrument for phytochrome assay. including dur- ing initial isolation and purification procedures. Using the Cary 118C spectrophotometer in conjunction with the dif- ference spectra device attachment. we have been able to measure phytochrome level down to ca. 1 pg,m/. since the spectrophotometer can readily record AA = 5 x lo-‘. Spruit (1970) also described a modification of the Cary-14 spectrophotometer for actinic irradiation from the same direction as the measuring beam. However. our modifica- tion is simpler. is achieved economically. and provides excellent reproducibility.

It should be emphasized that the two cells used for double difference spectra must be optically well matched and cleaned. However. since the cuvettes containing phyto- chrome are always kept in the same position and the double difference spectra are measured by simply switching the filters. some of the optical artifacts are cancelled out.

I I I I I

Wavelength, nm 600 700 800

Figure 3. The absorption (top panel) and double difference (lower panel) spectra of crude extracts from etiolated oat

seedlings in - 50 mM Tris buffer (pH 7.8).

DESIGN OF DOUBLE DIFFERENCE SPECTRA ATTACHMENT

The double difference spectra attachment is shown in Fig. 2. Essentially identical devices can be economi- cally built (total cost including light source and filters < $200) to fit the cell compartments of other spectro- photometers.

The actinic light is supplied by a 60W Westing- house light bulb of the show-case type or any other lamps of cylindrical envelope. The 660 (ca. 10nm band width: 0.27 W,m2) and 720 nm filters (plus IR filter: 1.8 W/m2) are fitted into two holes of 3.8 cm diameter so that the sample cuvettes are centrally positioned under the filters. The device shown in Fig. 2 completely blocks the actinic light between the two cell compartments to achieve independent actinic irradiation of each cuvette.

Acknowledgeri7e,it~-This work was supported by the National Science Foundation (PCM75-05001 -A01) and the Robert A. Welch Foundation (D-182). The advice of Dr. L. H. Pratt regarding phytochrome isolation is greatly appreciated.

REFERENCES

Butler. W. L. (1964) Ann. Rer. Plan1 Phjxiol. 15, 451-470. Butler. W. L.. S. B. Hendricks and H. W. Siegelman (1964) Phorochem. Photobiol. 3, 521-528.

Technical note 42 I

Butler. W. L.. K. H. Norris. H. W. Siegelman and S. B. Hendricks (1959) Proc. Nut/ . Acud. Sci

Furuya, M. (1976) Phytochrome (in Japanese). pp. 87-95. Iwanami. Tokyo. Kendrick. R. E. and C. J. P. Spruit (1977) Photochrm. Phorohiol. 26. 201 -214. Kidd. G. H. and Pratt, L. H. (1973) Pltrnt Physiol. 52, 309-311. Pratt. L. H. (1978) Photoclrrm. Photohiol. 27, 81-105. Pratt. L. H. and R. A. Coleman (1971) Proc. N d . Acud. Sci. U.S. 68, 2431-2435. Pratt. L. H. and D. Marme (1976) Plant Physiol. 58, 686-692. Spruit. C. J. P. (1970) Medrd. Landhouwhog. Wagcwingcv 70-140, 1-8. Tobin. E. M. and W. R. Briggs (1973) Photochurn. Phorohiol. 18, 487-498.

U.S. 45, 1703-1708.