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Identification of the blue-fluorescent compounds in boron-deficient plants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
LETTERS
Identification
of the Blue-Fluorescent
64, 506-516 (1956)
TO THE EDITORS
Compounds
in Boron-Deficient
Plants’
Spurr (1) has reported a blue fluorescence in the necrotic areas of boron-deficient celery leaves and petioles. Our investigations have shown that a blue fluorescence is also present in and around the necrotic areas on the leaves of boron-deficient tomato, lettuce, radish, and sunflower plants. That the fluorescence results from chlorogenic and caffeic acids has been shown in the following manner. Corresponding leaves from boron-deficient and healthy plants were ground at room temperature with 80% ethanol in glass homogenizer tubes. Followingcentrifugation of the homogenate, the supernatant was extracted with petroleum ether, concentrated under reduced pressure, and chromatographed ascendingly on Whatman No. 1 filter paper in eight solvent systems. While at least four blue-fluorescent spots were noted on most of the chromatograms, two of these spots were very much larger in the case of the boron-deficient, as compared to the healthy, leaves. Elution of these spots and co-chromatography with authentic chlorogenic and caffeic acids resulted in complete coincidence in all solvents. Treatment of the presumed chlorogenic acid with 2 N HCl at 100°C. for I hr. gave caffeic acid, quinic acid, and a little residual chlorogenic acid. The presumed caffeic acid showed no apparent change on similar treatment. Authentic samples of chlorogenic and caffeic acids gave results identical to those obtained for the unknown compounds. The fluorescence of the spot corresponding to chlorogenic acid changed from blue to bright green on exposure to ammonia vapor while the presumed caffeic acid became a more intense blue, both observations being in accord with the reactions of authentic material. The presumed chlorogenic acid spot after exposure to ammonia vapor was yellow by daylight. Spraying of the spots with 2 N iVaOH, ammoniacal silver nitrate, diazotized p-nitroaniline (with and without an overspray of sodium carbonate solution), diazotized sulfanilic acid, and ferric chloride solution (2) produced colors which were in all cases entirely consistent with their identification as chlorogenic and caffeic acids. A much higher concentration of these phenolic acids was present in the green leaf material immediately adjacent to t,he necrotic area than in either the rest of the leaf or the necrotic area itself. This observation and the fact that both chlorogenie and caffeic acids were found (in lesser amounts) in healthy leaves suggests that the formation of blue-fluorescent areas in the leaves of boron-deficient plants 1 This work was supported in part by contract with the Atomic Energy Commission. Contribution number 463 from the Inst,itute for Atomic Research and the Botany Department, Iowa State College, Ames, Iowa. 506
LETTERS
TO
THE
507
EDITORS
is a secondary effect of the deficiency. Since the amounts of these acids present in the necrotic area of the leaf were found to be much less than those in the green part adjacent to the necrosis, the chlorogenic and caffeic acids may be condensed by polyphenoloxidase to melanin pigments.
1. SPURR, A. R., Science 116, 421 (1952). 2. SWAIX, T., ~iochem. J. 63, 200 (1952). Znstitute for Atomic Research and Botany Zowa Slate College, Ames, Zowa Received July 24, 1956
On the Spontaneous
HAROLD J. PERKIXS S. ARONOFP
Department,
Aggregation
of Myosinl
At present there are in the literature some six values for the molecular weight of myosin ranging from 15 X 1Oj to 4 X 105. Laki and Carroll (1) have ascribed the differing values to the fact that myosin is altered considerably by exposure to room temperature. In the ultracentrifuge at 5°C. they observed a small leading peak produced a and a sharp major peak. Two hours’ exposure at room temperature single, very broad, asymmetric, more rapidly sedimenting peak. Whether this involves changes in shape, size, and/or aggregation of the molecules was not determined. We present here a brief account of experiments that help clarify this point.
a
b
FIG. 1. 0.36% myosin in 0.6 M KC1 at 25”C., centrifuged at 59,780 r.p.m. Fresh myosin; 51 min., (b) after standing 45 hr. at 25°C.; 46 min. 1 Contribution No. 1387 Sterling Haven, Connecticut.
Chemistry
Laboratory,
Yale University,
(a) New
OF
BIOCHEMISTRY
AND
BIOPHYSICS
LETTERS
Identification
of the Blue-Fluorescent
64, 506-516 (1956)
TO THE EDITORS
Compounds
in Boron-Deficient
Plants’
Spurr (1) has reported a blue fluorescence in the necrotic areas of boron-deficient celery leaves and petioles. Our investigations have shown that a blue fluorescence is also present in and around the necrotic areas on the leaves of boron-deficient tomato, lettuce, radish, and sunflower plants. That the fluorescence results from chlorogenic and caffeic acids has been shown in the following manner. Corresponding leaves from boron-deficient and healthy plants were ground at room temperature with 80% ethanol in glass homogenizer tubes. Followingcentrifugation of the homogenate, the supernatant was extracted with petroleum ether, concentrated under reduced pressure, and chromatographed ascendingly on Whatman No. 1 filter paper in eight solvent systems. While at least four blue-fluorescent spots were noted on most of the chromatograms, two of these spots were very much larger in the case of the boron-deficient, as compared to the healthy, leaves. Elution of these spots and co-chromatography with authentic chlorogenic and caffeic acids resulted in complete coincidence in all solvents. Treatment of the presumed chlorogenic acid with 2 N HCl at 100°C. for I hr. gave caffeic acid, quinic acid, and a little residual chlorogenic acid. The presumed caffeic acid showed no apparent change on similar treatment. Authentic samples of chlorogenic and caffeic acids gave results identical to those obtained for the unknown compounds. The fluorescence of the spot corresponding to chlorogenic acid changed from blue to bright green on exposure to ammonia vapor while the presumed caffeic acid became a more intense blue, both observations being in accord with the reactions of authentic material. The presumed chlorogenic acid spot after exposure to ammonia vapor was yellow by daylight. Spraying of the spots with 2 N iVaOH, ammoniacal silver nitrate, diazotized p-nitroaniline (with and without an overspray of sodium carbonate solution), diazotized sulfanilic acid, and ferric chloride solution (2) produced colors which were in all cases entirely consistent with their identification as chlorogenic and caffeic acids. A much higher concentration of these phenolic acids was present in the green leaf material immediately adjacent to t,he necrotic area than in either the rest of the leaf or the necrotic area itself. This observation and the fact that both chlorogenie and caffeic acids were found (in lesser amounts) in healthy leaves suggests that the formation of blue-fluorescent areas in the leaves of boron-deficient plants 1 This work was supported in part by contract with the Atomic Energy Commission. Contribution number 463 from the Inst,itute for Atomic Research and the Botany Department, Iowa State College, Ames, Iowa. 506
LETTERS
TO
THE
507
EDITORS
is a secondary effect of the deficiency. Since the amounts of these acids present in the necrotic area of the leaf were found to be much less than those in the green part adjacent to the necrosis, the chlorogenic and caffeic acids may be condensed by polyphenoloxidase to melanin pigments.
1. SPURR, A. R., Science 116, 421 (1952). 2. SWAIX, T., ~iochem. J. 63, 200 (1952). Znstitute for Atomic Research and Botany Zowa Slate College, Ames, Zowa Received July 24, 1956
On the Spontaneous
HAROLD J. PERKIXS S. ARONOFP
Department,
Aggregation
of Myosinl
At present there are in the literature some six values for the molecular weight of myosin ranging from 15 X 1Oj to 4 X 105. Laki and Carroll (1) have ascribed the differing values to the fact that myosin is altered considerably by exposure to room temperature. In the ultracentrifuge at 5°C. they observed a small leading peak produced a and a sharp major peak. Two hours’ exposure at room temperature single, very broad, asymmetric, more rapidly sedimenting peak. Whether this involves changes in shape, size, and/or aggregation of the molecules was not determined. We present here a brief account of experiments that help clarify this point.
a
b
FIG. 1. 0.36% myosin in 0.6 M KC1 at 25”C., centrifuged at 59,780 r.p.m. Fresh myosin; 51 min., (b) after standing 45 hr. at 25°C.; 46 min. 1 Contribution No. 1387 Sterling Haven, Connecticut.
Chemistry
Laboratory,
Yale University,
(a) New