Fraction I protein concentration in plants by analytical ultracentrifuge

ANALYTICAL

BIOCHEMISTRY

Fraction

66, 12-17 (1975)

I Protein by Analytical

Concentration Ultracentrifuge

J. M. MCARTHUR

in Plants

AND M. HIKICHI

Research Sfation, Agriculture Canada, Summerland, British Columbia, Canada, VOH

IZO

Received April 15, 1974; accepted November 11, 1974 The fraction I protein content of plants was determined by centrifuging extracts in the analytical ultracentrifuge until the protein boundary was well separated. The refractive index increase across the boundary was determined with the interference optical system and converted to fraction I protein concentration with the factor 0.245 mg/ml/fringe.

The fraction I protein in plants has a sedimentation coefiicient of 18s which is intermediate between and well separated from the ribosomes, 70s and 8OS, and the other (fraction II) proteins, 4-6s (1,2). In a plant extract centrifuged at high speed until the ribosomes are deposited on the cell bottom, the fraction I protein is in the lower part of the cell. Above the fraction I boundary the solute is the fraction II proteins and the soluble components of the extract. Below the boundary the solute is the same plus fraction I protein; the difference between the two is the fraction I protein. Refractive index is linearly related to concentration (3) and, therefore, the index increment across the boundary is a measure of the fraction I protein concentration. The increment can be measured in the analytical ultracentrifuge with the Rayleigh interference optical system (4,5). The increment as interference fringes can be converted to conventional weight/volume units by means of the refractive index or by calibrating with a standard protein (3). This paper describes the procedure we used to determine the fraction I protein in various plants (6,7). METHODS

Plant samples were stored at -20°C in closed polyethylene bags if they were to be analyzed later. Both frozen and fresh samples were ground with liquid nitrogen in a grinding mill (Fisher Scientific Co. 8-450) driven at 35 rpm. The ground material was mixed by the method of Hikichi and Miltimore (8) and 1 Contribution No. 387 from the Research Station, Agriculture British Columbia. 12 Copyright All rights

@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

Canada, Summerland,

PROTEIN

CONCENTRATION

IN

PLANTS

13

placed in an aluminum foil tray. The tray was then placed in a loosely closed polyethylene bag until the liquid nitrogen had volatilized. Before the sample thawed, the bag was closed tightly to prevent dehydration and stored at -20°C. A l-g aliquot of the frozen, ground material was ground in a Dual1 tissue grinder (Kontes Glass Co., Vineland, NJ K-885450, Size 23), with 2.5 ml 0.1 M phosphate buffer, pH 7.8, then centrifuged at 32,000g for 20 min. The supernatant fluid was retained for the analytical centrifugation. For dry weight a second aliquot was dried at 105°C for 24 hr. For the analytical ultracentrifuge run, 0.43 ml of the supematant fluid was placed in the right sector of a 12-mm double-sector interference cell with sapphire windows. The left sector contained 0.45 ml 1,3-butanediol solution whose refractive index was approximately 0.001 less than the sample solution. Five filled cells were loaded into an Analytical-G titanium rotor and centrifuged at 47,660 rpm for 32 min in a Beckman Model E analytical ultracentrifuge with the interference optical system. Interference patterns were photographed with Kodak Spectroscopic II-G plates. The fringe shift and the radial distances to the meniscus and the fraction I protein boundary were measured with a microcomparator (Nikon Shadowgraph Model 6C). The interference fringe shift was corrected for radial dilution by multiplying by the ratio of the square of the boundary radial distance to the square of the meniscus radial distance (5).

The relation of fringe shift to protein concentration was determined with solutions of bovine serum albumin (Sigma 905-10) in synthetic boundary cells (3). RESULTS

AND

DISCUSSION

Although there are differences in amino acid composition and various properties of fraction I proteins (9,15,20,22) the refractive indices are almost constant (Table 1). Further, they agree quite well with the index 1.60 ?Z 2% for simple proteins (3,23). Only two of the fraction I protein indices, spinach (15), and tobacco (15) are slightly higher than the protein index range. However, three other spinach indices (12,14.16) and the tobacco index calculated from later data (20) fall within the range. Because the refractive index of bovine serum albumin agrees with that for fraction I protein, within 0.5% (3), it is stable and readily available in good quality, we used it for calibrating. The mean of our determinations differed by only 0.4% from fraction I protein values (Table 2) indicating that is was satisfactory. In a multicomponent plant extract the Johnston-Ogston effect (25) could produce a reduced refractive increment at the fraction I protein

14

MCARTHUR

AND HIKICHI

TABLE 1 REFRACTIVE INDICES OF FRACTION I PROTEINS Plant

Data source (see references)

Refractive indexa

(10) (11)

1.603 1.613 1.608b 1.606 1.618 1.641 1.60@ 1.611b 1.605 1.605 1.598 1.636 1.622 1.6oob 1.62W 1.61@ 1.5940 1.612 k .0125

Alfalfa (Medicago sativa) Alga (Chalmydomonas reinhardi) (Chorilla

(12)

elliposoidea)

Oat (Avena sat&a) Spinach (Spinacia oleracea)

(13) (14) (15)

(16) (12) Spinach beet (Beta vulgaris Tobacco (Nicotiana (N. (N. (N. (N.

var. Cicla)

(17) (18) (19) (15)

tabacum)

(20) (20) (W (20) (20)

glutinosa) glum) rustica) sylvestris)

a Calculated from amino acid analysis by the method of McMeekin et al. (21). b It was assumed that the fraction I protein molecule consists of eight large and six small subunits (22).

boundary. To test for this effect we ground alfalfa leaves, because of their high protein content, in 1.5 ml phosphate buffer in place of the usual 2.5 ml. The extracts and dilutions were run in the analytical ultracentrifuge. The fringe shift was plotted against dilution and the curves extrapolated to zero concentration (Fig. 1). The plots indicated that the Johnston-Ogston effect was negligible. The fraction I protein can also be determined with other rotors or with the schlieren optical system. An aluminum rotor with the same configuTABLE PROTEIN

CONCENTRATION

INCREMENT

2 PER INTERFERENCE

FRINGE

SHIFT

Material

Data source

Concentration increment (mg/ml/fringe)

Proteins Proteins Fraction I proteins Bovine serum albumin Bovine serum albumin

(23) (3) Table 1 (24) Our data

0.245 0.244 f 0.0077 0.246 f 0.0019 0.244 0.245 + 0.0024

PROTEIN

CONCENTRATION

IN PLANTS

1.5

18,

RELATIVE

CONCENTRATION

FIG. I. Test for Johnston-Ogston effect: number of interference fringes versus dilutions of alfalfa extracts, 1.5 ml 0.1 M phosphate buffer, pH 7.8, per g leaves.

ration as the titanium rotor used in this work gives equally satisfactory results but requires approximately 12 min longer per run. With the schlieren optics, only two samples can be analyzed per run and the refractive increment is calculated from the areas under the peaks: a slower and less accurate procedure than counting fringes in the interference method. Fraction I protein is denatured at pH below 6.5 (1) and by foaming (2). A low pH can be prevented during extraction with buffer solutions or by adding alkaline salts to the sample but the extracts cannot be stored for long without protein loss. Foaming can be a problem during comminution particularly in samples containing fiber. Both these problems can be circumvented by grinding with liquid nitrogen or solid carbon dioxide. Although freezing denatures fraction I protein in solution it does not in plant tissue and there is no difference in the recovery from fresh and frozen leaves. In our experience, the ground samples stored at -20°C are stable for at least a year but if allowed to thaw there is a rapid loss of fraction I protein unless immersed in a buffer which will keep the pH above 6.5. Some plants contain tannins (26) which precipitate proteins during sample preparation. This loss can be prevented by adding to the buffer

16

MCARTHUR

THE EFFECT

OF

AND

HIKICHI

TABLE 3 PVP ON THE EXTRACTION FROM

VARIOUS

OF FRACTION PLANTS

I PROTEIN

Fraction I protein (% oven dry weight) Plant Astrolagus Desmodium Lespedeza

ricer intortum unicatum cuneata (high

stipulacea Lotonolis bainsai Lotus corniculatus

pedunculatus Lupinus albus Medicago sativa Onobrychis viciaefolia

Sanguisorba minor Trifolium arvense fragiferam hybridium incarnatum medium pratense

Vicia

saliva villosa

tannin selection) (low tannin selection)

‘Cascade’ ‘Empire’ ‘Leo’

‘Eski’ ‘Krasnodor’ ‘Melrose’ ‘Visnorsky’ ‘Viva’

‘Mammoth Red’ ‘Medium’ ‘Pennscott’

0% PVP

2% PVP

3.06 0 0.57 0 2.13 0.78 3.79 0.88 2.35 2.10 0 4.64 5.32 0 0 0 0 0 0 0 2.57 3.79 2.32 2.93 4.94 4.50 4.31 3.22 4.06

3.03 4.25 4.99 3.14 2.68 3.04 3.62 2.28 2.28 2.43 2.54 4.78 5.37 4.22 4.41 4.25 3.85 4.41 1.71 1.66 2.58 3.45 2.07 3.08 4.88 4.58 4.37 3.25 4.14

solution 2% polyvinylpyrrolidone (Badische-Anilin-und Soda-Fabrik AG, Kollidon 25) (Table 3). This requires about 10 min extra centrifugation because of the lower sedimentation rate in the higher viscosity solutions. Lower-strength phosphate and other buffer mixtures have been used with equally good results. However, at times, there was some loss of protein if the extract was below pH 6.5. We have not had any problems with the 0.1 M phosphate, pH 7.8, buffer.

PROTEIN

CONCENTRATION

IN PLANTS

17

REFERENCES 1. Kawashima, N., and Wildman, S. G. (1970) Annu. Rev. Plnnt Physiol. 21, 325-358. 2. McArthur, J. M., Miltimore, J. E., and Pratt, M. J. (1964) Can. J. Anim. Sci. 44, 200-206. 3. Babul, J., and Stellwagen, E. (1969) Anal. Biochem. 28, 216-221. 4. Schachman, H. K. (1959) Ultracentrifugation in Biochemistry, Academic Press, New York. 5. Chervenka, C. H. (1970) A Manual of Methods for the Analytical Ultracentrifuge, Spinco Division of Beckman Instruments, Inc., Palo Alto, CA. 6. Heinrichs, D. H., and Miltimore, J. E. (1970) Can. J. Plan? Sci. 50, 537-539. 7. Miltimore, J. E., McArthur, J. M., Goplen, B. P., Majak, W., and Howarth, R. E. (1974) Agron. J. 66, 384-386. 8. Hikichi, M., and Miltimore, J. E. (1970) Lab. Pratt. 19, 383. 9. Kawashima, N., Imai, A., and Tamaki, E. (1968) Agr. Biol. Chem. 32, 535-536. 10. McArthur, J. M. Unpublished data. 11. Givan, A. L., and Criddle, R. S. (1972) Arch. Biochem. Eiophys. 149, 153-163. 12. Sugiyama, T., Ito, T., and Akazawa, T. (1971) Biochemistry 10, 3406-3411. 13. Steer, M. W., Gunning, B. E. S., Graham, T. A., and Carr, D. J. (1968) Planfa (Berlin) 79, 254-267. 14. Rutner, A. C., and Lane, M. D. (1967) Biochem. Biophys. Res. Commun. 28, 531-537. IS. Kawashima, N. (1969) Plant Cell Physiol. 10, 3 I-40. 16. Sugiyama. T., and Akazawa, T. (1970) Biochemistry 9,4499-4504. 17. Thornber, J. P., Ridley, S. M., and Bailey, J. L. (1965) Eiochem. J. 96, 29C-31C. 18. Ridley. S. M., Thomber, J. P.. and Bailey, J. L. (1967) Biochim. Biophys. Acta 140, 62-79. 19. Moon, K. E., and Thompson, E. 0. P. (1969) Aust. J. Biol. Sci. 22, 463-470. 20. Kawashima, N., Kwok, S., and Wildman, S. G. (1971) Biochim. Biophys. AC~U 235, 578-586. 2 1. McMeekin. T. L., Wilensky, M., and Groves, M. L. (1962) Biochem. Biophys. Res. Commun. I, 151-156. 22. Kawashima, N. (1971) Tampakushitsu Kakusan, KOSO~, 865-873. 23. Doty, P.. and Geiduschek, E. P. (1953) in The Proteins (Neurath, H., and Bailey, K.. eds.), Vol. lA, pp. 363-460, Academic Press, New York. 24. Instruction Manual Beckman Model E Ultracentrifuge E-lM-3 (1966), Spinco Division of Beckman Instruments, Inc., Palo Alto, CA. 25. Johnston, J. P., and Ogston, A. G. (1946) Trans. Faraday SOC. 42, 789-799. 26. Haslam. E. (1966) Chemistry of Vegetable Tannins, Academic Press, London,