- Email: [email protected]
Biosynthesis of carotenoids in Neurospora. Action spectrum of photoactivation
Biosynthesis of Carotenoids in Neurospora. Action Spectrum of Photoactivation Marko Zalokar’ From the National
Institute of Arthritis and Metabolic Diseases, Institutes of Health, Bethesda, Maryland Received
October
Xational
4, 1954
In his early research on Neurospora, Went (1,2) noted that light stimulated the production of carotenoids. Went (2) and Haxo (3), by using colored filters, found that the active part of the spectrum was in the blue region: red light was not active. Van Deventer (4) has shown that the effective wavelengths were contained in a spectral region extending from 510 to 366 mp. The light effect could be better observed on the mycelium, which remained nearly colorless in the dark, than on the conidia which, without illumination, produced considerable amounts of pigment. In a previous paper (5) a method was described for preventing conidiation in cultures of Neurospora by adding polyoxyethylene sorbitan monooleate (Tween SO) to the medium. This made it possible to measure the light effect more accurately and to determine the action spectrum. The action spectrum might indicate which substance serves as the light absorber in the process. Besides being of interest as an indicator of the pathways of carotenoid biosynthesis, study of the photosensitive substance would aid in the understanding of other photosensitive process in plants. METHODS The methods of growth and measurement of carotenoids in the Neurospora mycelium were described previously (5). The strain derived from a cross of wild types E 5297a and E 52568 was’used. The cultures were grown in 125-ml. Erlenmeyer flasks on minimal medium with Tween 80 added, for 3 days in the dark at 35°C. The mycelium pads (approximate dry weight 100 mg.) were filtered and exposed to light at room temperature under the given conditions. They were then 1 Present
address:
Wesleyan
University, 318
Middletown,
Connecticut.
BIOSYNTHESIS
OF
319
CAROTENOIDS
kept in the dark for 6 hr., and the carotenoids were extract.ed with methanol and acetone. Extracts of the individual mycelia were collected in 25-ml. volumetric flasks, and the light absorpCon was read with a Beckman spectrophotometer at 470 mp. The spectrographic work was performed at the U. S. Department of Agriculture Plant, Industry St,ation, Beltsville, Maryland. The spectrograph (6) with its greatf dispersion at, high intensity allowed t,he irradiation of the mycelia inside narrow spectral bans. Round mycelial pads, 5 cm. wide, were lined up on the projected image of the spectrum. Each pad was exposed to a band varying from 8 5 111~ wide at 500 mp to 2.5 mp wide at 400 mM. EXPERIMENTAL
Light Fffect In the preliminary studies the response to different illumination times of white light, was measured. The constant light sourvc was a fluorescent daylight desk-type lamp (one Is-W. tube) at a distance of 70 cm. The a.mount of pigment produced was directly proportional to the amount of illumination at the lower dosages (Fig. 1). In order t,o determine whether the photoeffert in Neurospora followed 200
I SC
5 c u z F x w
100
01 0
I 2
I 4
I 6
I 8
I IO
I 12
I 14
16
MINUTES
FIN. 1. Total carotenoid production by Neurospora as a function of light dosage. The mycelia were irradiated for increasing times, and 6 hr. later the carotenoids were extracted and the optical extinction measured at 470 rnp.
320
MARK0
01 70 5
ZALOKAR
141 DISTANCE
200
282
FIG. 2. Reciprocity of light effect. The abscissa indicates distances from the light sources in centimeters, corresponding light intensities being 1, >/4, 36, Ms. The ordinate shows the optical extinction values of the carotenoids produced. The exposure time was increased with distance to give the same light dosages. The exposure in curve A varied from 1 to 16 min.; in curve I3 from 2 to 32 min.; in curve C from 4 to 64 min.; and in curve D from 8 min. to 2 hr. 8 min.
the reciprocity law, the mycelia were exposed at 70.5, 141, 200, and 282 cm. from a point source of light obtained from an unshielded 6-v. automobile light bulb (as used in microscope lamps), which represented relative light intensities of 1, x, 46, and Ms. The results (Fig. 2) when plotted for equal dosages and different exposures (or intensities) did not show a complete reciprocity. Even when a correction for the fact that the light source was not an ideal point was attempted, the more distant mycelia (needing longer exposures) showed a higher response than the mycelia receiving the same dosage in a shorter time, i.e., for a constant dosage, a longer exposure was more effective. The action spectrum was determined by placing the mycelia in the spectrum and exposing them to equal total energies of radiation. The quantity of pigment produced in the next 6 hr. in the dark was recorded. The energies were calculated in order to give about half the maximum response (pigmentation) at the most effective wavelength (465 mp), which amounted to lo6 ergs/sq. cm. The intensity of the spectrum decreased about fivefold between 500 and 400 rnp. To obtain equal dosages, the exposure times were varied from 2 to 6 min., and at higher intensities the light was screened with a 43 % filter. As shown from reciprocity ex-
BIOSYNTHESIS
OF
321
CAROTENOIDS
llll1llllll
I
I
I
I
I
I
.lZO-
.I00
3
k5080
-
F s!
GO60
l
4
-
I-
1:
0
400
“II”“‘II”
, 410 , 420 439 , 430, , 449 405 415 425 434 444 WAVELENGTH
, 454
I
”
473
460 4&
II
400 4Lo
500 4k5
M/i
FIG. 3. Action spectrum of carotenoid production in it’ezcrospo,n.All stations (Lbeir centers indicated in the abscissa) were irradiated witA equal energies of light,. Carotenoids were extracted 6 hr. later, and the optical cstinction was
measured
at 470 mp.
periments, the longer exposure t,imes required at shorter n-avelengths should result in a relatively higher response; on the other hand, a (‘orrection for the number of quanta, which is lower at t,he shorter wavelengt,hs for t,he same energy, would give lower vahles. These differences were not large enough to change the results materially. The curve of the action spectrum (Fig. 3) presents measurements from four experiments performed on the same day and one at, another time. The limitations of the spectrograph prevented the use of radiation shorter than 400 mp. The activity increased slowly from 400 to 449 mp. Thrrc was a plateau between 449 and 488 rnp in which analysis of variance did not reveal any parbicular peak, although the mean values were highest at 480 rnp. Activity dropped sharply between 490 and 510 rnp (n-hen ten Cmes higher energies were used, the activity reased at 520 mp). No effect was observed in the red side of t,he spectrum even after 45 min. of cxposure. Yellow Pigments Although the mycelial pads appear white before exposure, they must contain traces of a pigment with the absorption range of the action spec-
322
MARK0
ZALOKAR
trum, which is responsible for the light effect. In a previous publication (5), we have reported all the carotenoids found in these mycelia. The main polyenes were the colorless, ultraviolet-absorbing phytoene and phytofluene. Small quantities of {-carotene, and even less neurosporene, y-carotene, and spirilloxanthin could be observed. The acidic pigment and j3-carotene could not be detected in the extracts. The alcohol-acetone extracts, from which the carotenoids were separated by shaking wit.h hexane, conta.ined a red-yellow pigment which could be ext.racted with ether from a concentrated solution. Its sharp absorption peak at 408 rnp suggested a pigment of the porphyrin type. The extra&on of the mycelium with ice-cold water gave a yellowish solution. After passing this solution through a magnesium silicate adsorption column, flavins and a red-yellow pigment of unknown composition were adsorbed. They could be developed and eluted with 5% acetone. The unknown pigment had an absorption maximum at 410 mp. The unadsorbed fraction could he concentrated and proteins and salts precipitated uit.h the addition of alcohol. The yellow supernatant showed an absorption which increased uniformly from 520 mp to a shallow peak at 420 rnp. Other yellow fractions Were obtained by extracting the mycelium wit,h hot water and using Dowex 1 or Dowex 50 ion-exchange resins. Except for Aavin derivatives, all these fractions showed steadily increasing absorption from green to blue and had their main absorption peaks in the ultraviolet part of the spectrum. Of all the water-soluble pigments, only riboflavin and its derivatives showed light-absorption curves similar t,o that of the action spectrum. The total amount of riboflavin in the mycelium was measured fluorometrically, after hydrolyzing t,he mycelium in 0.1 N HCl and using the method of Loy (7). The average amount found in each mycelium pad was 4.45 pg. (41 pg./g. dry weight of mycelium). T\‘o difference was observed between illuminated and nonilluminated mycelia. Probably only part of this riboflavin existed in the mycelium in the free state, the rest of it being present in the combined form as the prosthetic group of enzymes. Temperature Coeficient The over-all photochemical reaction in the product,ion of carotenoids was temperature sensitive and dropped pract.ically to zero at 0°C. The primary reaction, however, was independent of temperature, which con-
BIOSYNTHESIS OF CAROTENOTDS
323
firmed earlier observations recorded by Haxo (8). The mycelial pads were cooled to O”C., kept at room temperature, or warmed to 37°C. They were then irradiated with a dosage which gave one-half response at room temperature; after irradiation at the three temperatures they were kept in t,he dark at 25°C. for A hr. There were no significant diffcrewes in the amount of pigment produced. The mycelia exposed at 0°C. could be kept in the dark at that temperature for 24 hr. wit#hout pigment,atSion; if they were then warmed to 25”C., t)hey formed as much pigment as if they had been warmed immediat’ely aft.er exposure. DISCUSSION
The evaluation of the action spectrum was made on the assumption that it indicates the absorption range of the substance which serves as the light receptor, while recognizing the possibility that a quantum energy threshold could limit the action spectrum on the red side. In Nezcrosporu, only riboflavin derivatives could be considered as the phot,oreceptor, since no other pigment present was observed to cover the same range of the spectrum. Most of t,he carotenoids absorbed light over wider ranges, or t,heir maxima were significantly to the left or to t,he right of the acticon spectrum. P-Carotene covers the observed range, but its presence could not be detected in the unexposed mycelium, this pigment being formed only at a later time after exposure to light (5). Riboflavin itself could not be the photoreceptor, since its spectrum has a range about 20 rnp closer to the blue side than the action spectrum. Riboflavin is part, of the prosthetic groups of several enzymes (flaroproteins) and is responsible for t*heir light absorption in t.he visible spectrum. Combining with proteins shift’s the riboflavin spectrum, usually toward the red. The “old” yellow enzyme of Warburg had a maximum absorption at 465 rnp and a minimum at 415 ml, and the absorption increased toward another peak at 380 rnp (9), thus covering closely the range of the action spectrum. The increase near the ultraviolet was not observed in the action spectrum, the only fact which does not seem to be in favor of flavoproteins. Too much weight should not be given to this observation because of increased light watt’ering at lower wavelengths, the possible screening effect of other pigmems, and inadequacies of the experiment)al method. Riboflavin was considered as a probable receptor in several light-sensitive reactions in organisms (10) and was proved to be responsible for the photochemical destruction of indoleacetic acid (auxin).
324
MARK0
ZALOKAR
It is known that the photoactivation is a complex reaction involving several steps (11). The primary effect of the absorbed light quantum is to activate the absorbing molecule. The activated molecule then transfers energy to, or interacts chemically with, some other molecule. If all the available energy is used in this process, no further changes occur as a direct effect of the absorption of one quantum. On the other hand, the activated molecule can induce an unstable state in the second molecule which may lead to a chain reaction. Such a reaction can affect many molecules, and as a result the quantum yield (number of molecules transformed for one quantum absorbed) can be very high. Both types of reactions would occur immediately after photoactivation, and their temperature coefficient would be close to one. These reactions can then produce the conditions for the inception of a new react,ion which can progress slowly in the dark and which has a high temperature coefficient. If we assume that all riboflavin in the mycelium participated (as a flavoprotein) in the photochemical reaction, we can calculate from the amount of riboflavin present the percentage of irradiation energy absorbed and thus the maximum possible number N (in moles) of molecules activated. The following formula was used: N=EXE’XA hv X M where E = incident energy (lo6 ergs/sq. cm.) ; E’ = the fraction of light absorbed by the pigment present (1.18 X Wa moles of riboflavin, with molecular absorption coefficient taken for a flavin (9) E= 1.04 X lo4 at 465 rnp; correction for reflected light taken as 50%, light screening by other pigments is negligible; which gives E’ = 0.57 %) ; A = area of mycelium exposed (25 sq. cm.) ; hv = quantum energy at 465 rnp (4.26 X 1O-*2);and M = Avogadro’s number. This gave 5.55 X 1O-9 moles of activated molecules. The carotenoids extracted from the mycelium gave an extinction of 0.120 in a 25-n-L sample, which corresponds to about 12 pg. (2.2 X lo-* moles) of pigment (5). This gave a minimum quantum yield of 4.0 molecules of carotenoids produced for one quantum absorbed. The actual quantum yield may be much higher since not necessarily all of the riboflavin is present in photochemically active form. The quantum yield was calculated for the over-all process of pigment formation, including the dark reaction. Since the quantum yield for the primary reaction is one, the photoactivated flavin could not have directly converted enough molecules into the precursors of carotenoids. There must have been a secondary reaction responsible for the quantum
BIOSYNTHESIS OF CAROTENOIDS
325
yield higher than one. The calculated quantum yield was not so high as to exclude the possibility that the photoactivated flavin initiated a chain reaction transforming the precursors of carotenoids. More probable would be the hypothesis that the photoactivated flavin activated (or reversed an inhibition of) some enzyme system which was necessary for the performance of the dark react~ion. The product,ion of carotenoids would then be proportional to the degree of activation and could amount to any multiple of the absorbed quanta. ACKNOWLEDGMENT The author wishes to express his deep gratitude to Dr. S. B. Hendricks for his permission and aid in the use of the spectrograph and for his advire during the course of t,his invcstigat’ion. SUMMARY
The amount of carotenoids produced in Neurospora was proportional to the light dosage. With longer exposure, the same dosage had a slightly higher effect. The temperature coefficient for the light effect, was one, but the subsequent, production of pigments was temperature dependent. The action spectrum was determined between 400 and 500 rnp. There was no light action beyond 520 mp. The action spectrum corresponded best to a spectrum of a riboflavin derivative, and no other pigmenhs with a similar spectrum could be detected in Neurospora. It was therefore assumed t’hat a flavin was bhe photoreceptor. There were at least four molecules of carotenoids produced for one quantum of light absorbed, as cbalculated from the light absorption by riboflavin present in t,he mycelinm. REFERENCES 1. WENT: F. .i. F. C., Cenfr. Rah2e~iol. Pnrcuiienk. (II) 7, 544 (1301). 2. WENT, F. A. F. C., Xev. Tram. Rotan. Neerl. l-4, 106 (1904). 3. H~xo. F , birch. Biochewl. 20, 400 (1949). 4. VAN DEVENTER, W. F., Thesis. TJtrecht, Holland, 1930. 5. ZALOKAR, &I., Arch. Biochem. 6. PARKER, hf. Iv., HENDRICKS,
and Bzophys. 60, il (1054). S. B., BORTHWICK, H. .4., AXD
SCULLY, N. F., Botan. Gaz. 108, 1 (1946). 7. GYDRC,T,I’., “Vitamin Metjhods,” Vol. II, p. 625. Academic Press Inc., New York, 1951. 8. HAXO, F., Dissertation. Stanford Univ., Stanford, Calif., 1947. !I. TIIEORELL, H., Biochem. 2. 278, 263 (1935). 10. GALSTOY: r\ W., Science 111, 619 (1950). 11 BLUM, H IT., “Photod~namic 11ction and Diseases C’auscxtl 1,~ l.ipht ” Reinhold PII~)I Corp., NenT York, 1941.
Institute of Arthritis and Metabolic Diseases, Institutes of Health, Bethesda, Maryland Received
October
Xational
4, 1954
In his early research on Neurospora, Went (1,2) noted that light stimulated the production of carotenoids. Went (2) and Haxo (3), by using colored filters, found that the active part of the spectrum was in the blue region: red light was not active. Van Deventer (4) has shown that the effective wavelengths were contained in a spectral region extending from 510 to 366 mp. The light effect could be better observed on the mycelium, which remained nearly colorless in the dark, than on the conidia which, without illumination, produced considerable amounts of pigment. In a previous paper (5) a method was described for preventing conidiation in cultures of Neurospora by adding polyoxyethylene sorbitan monooleate (Tween SO) to the medium. This made it possible to measure the light effect more accurately and to determine the action spectrum. The action spectrum might indicate which substance serves as the light absorber in the process. Besides being of interest as an indicator of the pathways of carotenoid biosynthesis, study of the photosensitive substance would aid in the understanding of other photosensitive process in plants. METHODS The methods of growth and measurement of carotenoids in the Neurospora mycelium were described previously (5). The strain derived from a cross of wild types E 5297a and E 52568 was’used. The cultures were grown in 125-ml. Erlenmeyer flasks on minimal medium with Tween 80 added, for 3 days in the dark at 35°C. The mycelium pads (approximate dry weight 100 mg.) were filtered and exposed to light at room temperature under the given conditions. They were then 1 Present
address:
Wesleyan
University, 318
Middletown,
Connecticut.
BIOSYNTHESIS
OF
319
CAROTENOIDS
kept in the dark for 6 hr., and the carotenoids were extract.ed with methanol and acetone. Extracts of the individual mycelia were collected in 25-ml. volumetric flasks, and the light absorpCon was read with a Beckman spectrophotometer at 470 mp. The spectrographic work was performed at the U. S. Department of Agriculture Plant, Industry St,ation, Beltsville, Maryland. The spectrograph (6) with its greatf dispersion at, high intensity allowed t,he irradiation of the mycelia inside narrow spectral bans. Round mycelial pads, 5 cm. wide, were lined up on the projected image of the spectrum. Each pad was exposed to a band varying from 8 5 111~ wide at 500 mp to 2.5 mp wide at 400 mM. EXPERIMENTAL
Light Fffect In the preliminary studies the response to different illumination times of white light, was measured. The constant light sourvc was a fluorescent daylight desk-type lamp (one Is-W. tube) at a distance of 70 cm. The a.mount of pigment produced was directly proportional to the amount of illumination at the lower dosages (Fig. 1). In order t,o determine whether the photoeffert in Neurospora followed 200
I SC
5 c u z F x w
100
01 0
I 2
I 4
I 6
I 8
I IO
I 12
I 14
16
MINUTES
FIN. 1. Total carotenoid production by Neurospora as a function of light dosage. The mycelia were irradiated for increasing times, and 6 hr. later the carotenoids were extracted and the optical extinction measured at 470 rnp.
320
MARK0
01 70 5
ZALOKAR
141 DISTANCE
200
282
FIG. 2. Reciprocity of light effect. The abscissa indicates distances from the light sources in centimeters, corresponding light intensities being 1, >/4, 36, Ms. The ordinate shows the optical extinction values of the carotenoids produced. The exposure time was increased with distance to give the same light dosages. The exposure in curve A varied from 1 to 16 min.; in curve I3 from 2 to 32 min.; in curve C from 4 to 64 min.; and in curve D from 8 min. to 2 hr. 8 min.
the reciprocity law, the mycelia were exposed at 70.5, 141, 200, and 282 cm. from a point source of light obtained from an unshielded 6-v. automobile light bulb (as used in microscope lamps), which represented relative light intensities of 1, x, 46, and Ms. The results (Fig. 2) when plotted for equal dosages and different exposures (or intensities) did not show a complete reciprocity. Even when a correction for the fact that the light source was not an ideal point was attempted, the more distant mycelia (needing longer exposures) showed a higher response than the mycelia receiving the same dosage in a shorter time, i.e., for a constant dosage, a longer exposure was more effective. The action spectrum was determined by placing the mycelia in the spectrum and exposing them to equal total energies of radiation. The quantity of pigment produced in the next 6 hr. in the dark was recorded. The energies were calculated in order to give about half the maximum response (pigmentation) at the most effective wavelength (465 mp), which amounted to lo6 ergs/sq. cm. The intensity of the spectrum decreased about fivefold between 500 and 400 rnp. To obtain equal dosages, the exposure times were varied from 2 to 6 min., and at higher intensities the light was screened with a 43 % filter. As shown from reciprocity ex-
BIOSYNTHESIS
OF
321
CAROTENOIDS
llll1llllll
I
I
I
I
I
I
.lZO-
.I00
3
k5080
-
F s!
GO60
l
4
-
I-
1:
0
400
“II”“‘II”
, 410 , 420 439 , 430, , 449 405 415 425 434 444 WAVELENGTH
, 454
I
”
473
460 4&
II
400 4Lo
500 4k5
M/i
FIG. 3. Action spectrum of carotenoid production in it’ezcrospo,n.All stations (Lbeir centers indicated in the abscissa) were irradiated witA equal energies of light,. Carotenoids were extracted 6 hr. later, and the optical cstinction was
measured
at 470 mp.
periments, the longer exposure t,imes required at shorter n-avelengths should result in a relatively higher response; on the other hand, a (‘orrection for the number of quanta, which is lower at t,he shorter wavelengt,hs for t,he same energy, would give lower vahles. These differences were not large enough to change the results materially. The curve of the action spectrum (Fig. 3) presents measurements from four experiments performed on the same day and one at, another time. The limitations of the spectrograph prevented the use of radiation shorter than 400 mp. The activity increased slowly from 400 to 449 mp. Thrrc was a plateau between 449 and 488 rnp in which analysis of variance did not reveal any parbicular peak, although the mean values were highest at 480 rnp. Activity dropped sharply between 490 and 510 rnp (n-hen ten Cmes higher energies were used, the activity reased at 520 mp). No effect was observed in the red side of t,he spectrum even after 45 min. of cxposure. Yellow Pigments Although the mycelial pads appear white before exposure, they must contain traces of a pigment with the absorption range of the action spec-
322
MARK0
ZALOKAR
trum, which is responsible for the light effect. In a previous publication (5), we have reported all the carotenoids found in these mycelia. The main polyenes were the colorless, ultraviolet-absorbing phytoene and phytofluene. Small quantities of {-carotene, and even less neurosporene, y-carotene, and spirilloxanthin could be observed. The acidic pigment and j3-carotene could not be detected in the extracts. The alcohol-acetone extracts, from which the carotenoids were separated by shaking wit.h hexane, conta.ined a red-yellow pigment which could be ext.racted with ether from a concentrated solution. Its sharp absorption peak at 408 rnp suggested a pigment of the porphyrin type. The extra&on of the mycelium with ice-cold water gave a yellowish solution. After passing this solution through a magnesium silicate adsorption column, flavins and a red-yellow pigment of unknown composition were adsorbed. They could be developed and eluted with 5% acetone. The unknown pigment had an absorption maximum at 410 mp. The unadsorbed fraction could he concentrated and proteins and salts precipitated uit.h the addition of alcohol. The yellow supernatant showed an absorption which increased uniformly from 520 mp to a shallow peak at 420 rnp. Other yellow fractions Were obtained by extracting the mycelium wit,h hot water and using Dowex 1 or Dowex 50 ion-exchange resins. Except for Aavin derivatives, all these fractions showed steadily increasing absorption from green to blue and had their main absorption peaks in the ultraviolet part of the spectrum. Of all the water-soluble pigments, only riboflavin and its derivatives showed light-absorption curves similar t,o that of the action spectrum. The total amount of riboflavin in the mycelium was measured fluorometrically, after hydrolyzing t,he mycelium in 0.1 N HCl and using the method of Loy (7). The average amount found in each mycelium pad was 4.45 pg. (41 pg./g. dry weight of mycelium). T\‘o difference was observed between illuminated and nonilluminated mycelia. Probably only part of this riboflavin existed in the mycelium in the free state, the rest of it being present in the combined form as the prosthetic group of enzymes. Temperature Coeficient The over-all photochemical reaction in the product,ion of carotenoids was temperature sensitive and dropped pract.ically to zero at 0°C. The primary reaction, however, was independent of temperature, which con-
BIOSYNTHESIS OF CAROTENOTDS
323
firmed earlier observations recorded by Haxo (8). The mycelial pads were cooled to O”C., kept at room temperature, or warmed to 37°C. They were then irradiated with a dosage which gave one-half response at room temperature; after irradiation at the three temperatures they were kept in t,he dark at 25°C. for A hr. There were no significant diffcrewes in the amount of pigment produced. The mycelia exposed at 0°C. could be kept in the dark at that temperature for 24 hr. wit#hout pigment,atSion; if they were then warmed to 25”C., t)hey formed as much pigment as if they had been warmed immediat’ely aft.er exposure. DISCUSSION
The evaluation of the action spectrum was made on the assumption that it indicates the absorption range of the substance which serves as the light receptor, while recognizing the possibility that a quantum energy threshold could limit the action spectrum on the red side. In Nezcrosporu, only riboflavin derivatives could be considered as the phot,oreceptor, since no other pigment present was observed to cover the same range of the spectrum. Most of t,he carotenoids absorbed light over wider ranges, or t,heir maxima were significantly to the left or to t,he right of the acticon spectrum. P-Carotene covers the observed range, but its presence could not be detected in the unexposed mycelium, this pigment being formed only at a later time after exposure to light (5). Riboflavin itself could not be the photoreceptor, since its spectrum has a range about 20 rnp closer to the blue side than the action spectrum. Riboflavin is part, of the prosthetic groups of several enzymes (flaroproteins) and is responsible for t*heir light absorption in t.he visible spectrum. Combining with proteins shift’s the riboflavin spectrum, usually toward the red. The “old” yellow enzyme of Warburg had a maximum absorption at 465 rnp and a minimum at 415 ml, and the absorption increased toward another peak at 380 rnp (9), thus covering closely the range of the action spectrum. The increase near the ultraviolet was not observed in the action spectrum, the only fact which does not seem to be in favor of flavoproteins. Too much weight should not be given to this observation because of increased light watt’ering at lower wavelengths, the possible screening effect of other pigmems, and inadequacies of the experiment)al method. Riboflavin was considered as a probable receptor in several light-sensitive reactions in organisms (10) and was proved to be responsible for the photochemical destruction of indoleacetic acid (auxin).
324
MARK0
ZALOKAR
It is known that the photoactivation is a complex reaction involving several steps (11). The primary effect of the absorbed light quantum is to activate the absorbing molecule. The activated molecule then transfers energy to, or interacts chemically with, some other molecule. If all the available energy is used in this process, no further changes occur as a direct effect of the absorption of one quantum. On the other hand, the activated molecule can induce an unstable state in the second molecule which may lead to a chain reaction. Such a reaction can affect many molecules, and as a result the quantum yield (number of molecules transformed for one quantum absorbed) can be very high. Both types of reactions would occur immediately after photoactivation, and their temperature coefficient would be close to one. These reactions can then produce the conditions for the inception of a new react,ion which can progress slowly in the dark and which has a high temperature coefficient. If we assume that all riboflavin in the mycelium participated (as a flavoprotein) in the photochemical reaction, we can calculate from the amount of riboflavin present the percentage of irradiation energy absorbed and thus the maximum possible number N (in moles) of molecules activated. The following formula was used: N=EXE’XA hv X M where E = incident energy (lo6 ergs/sq. cm.) ; E’ = the fraction of light absorbed by the pigment present (1.18 X Wa moles of riboflavin, with molecular absorption coefficient taken for a flavin (9) E= 1.04 X lo4 at 465 rnp; correction for reflected light taken as 50%, light screening by other pigments is negligible; which gives E’ = 0.57 %) ; A = area of mycelium exposed (25 sq. cm.) ; hv = quantum energy at 465 rnp (4.26 X 1O-*2);and M = Avogadro’s number. This gave 5.55 X 1O-9 moles of activated molecules. The carotenoids extracted from the mycelium gave an extinction of 0.120 in a 25-n-L sample, which corresponds to about 12 pg. (2.2 X lo-* moles) of pigment (5). This gave a minimum quantum yield of 4.0 molecules of carotenoids produced for one quantum absorbed. The actual quantum yield may be much higher since not necessarily all of the riboflavin is present in photochemically active form. The quantum yield was calculated for the over-all process of pigment formation, including the dark reaction. Since the quantum yield for the primary reaction is one, the photoactivated flavin could not have directly converted enough molecules into the precursors of carotenoids. There must have been a secondary reaction responsible for the quantum
BIOSYNTHESIS OF CAROTENOIDS
325
yield higher than one. The calculated quantum yield was not so high as to exclude the possibility that the photoactivated flavin initiated a chain reaction transforming the precursors of carotenoids. More probable would be the hypothesis that the photoactivated flavin activated (or reversed an inhibition of) some enzyme system which was necessary for the performance of the dark react~ion. The product,ion of carotenoids would then be proportional to the degree of activation and could amount to any multiple of the absorbed quanta. ACKNOWLEDGMENT The author wishes to express his deep gratitude to Dr. S. B. Hendricks for his permission and aid in the use of the spectrograph and for his advire during the course of t,his invcstigat’ion. SUMMARY
The amount of carotenoids produced in Neurospora was proportional to the light dosage. With longer exposure, the same dosage had a slightly higher effect. The temperature coefficient for the light effect, was one, but the subsequent, production of pigments was temperature dependent. The action spectrum was determined between 400 and 500 rnp. There was no light action beyond 520 mp. The action spectrum corresponded best to a spectrum of a riboflavin derivative, and no other pigmenhs with a similar spectrum could be detected in Neurospora. It was therefore assumed t’hat a flavin was bhe photoreceptor. There were at least four molecules of carotenoids produced for one quantum of light absorbed, as cbalculated from the light absorption by riboflavin present in t,he mycelinm. REFERENCES 1. WENT: F. .i. F. C., Cenfr. Rah2e~iol. Pnrcuiienk. (II) 7, 544 (1301). 2. WENT, F. A. F. C., Xev. Tram. Rotan. Neerl. l-4, 106 (1904). 3. H~xo. F , birch. Biochewl. 20, 400 (1949). 4. VAN DEVENTER, W. F., Thesis. TJtrecht, Holland, 1930. 5. ZALOKAR, &I., Arch. Biochem. 6. PARKER, hf. Iv., HENDRICKS,
and Bzophys. 60, il (1054). S. B., BORTHWICK, H. .4., AXD
SCULLY, N. F., Botan. Gaz. 108, 1 (1946). 7. GYDRC,T,I’., “Vitamin Metjhods,” Vol. II, p. 625. Academic Press Inc., New York, 1951. 8. HAXO, F., Dissertation. Stanford Univ., Stanford, Calif., 1947. !I. TIIEORELL, H., Biochem. 2. 278, 263 (1935). 10. GALSTOY: r\ W., Science 111, 619 (1950). 11 BLUM, H IT., “Photod~namic 11ction and Diseases C’auscxtl 1,~ l.ipht ” Reinhold PII~)I Corp., NenT York, 1941.