- Email: [email protected]
Raman and fluorescent emission of the human lens. A new fluorophor
Exp. Eye Res. (1978) 27, 737-741
LETTER
TO THE EDITORS
Raman and Fluorescent Emission of the Human Lens. A New Fluorophor The fluorescence of the human lens is a subject of widespread current interest because of its occurrence at levels elevated much above normal in nuclear senile cataracts (Augnsteyn, 1975; Lerman, Louis and Hollander, 1971; Pirie, 1968; Satoh, Bando and Nakajima, 1973; Spector, Roy and Stauffer; 1975; van Heyningen, 1973; Zigman, 1971). It has been proposed that the accelerated accumula,tion of fluorescent pigment is a manifestation of a polymerization which is part of the same reaction producing the pigment (Buckingham, 1952; Pirie, 1972; Truscott and Augusteyn: 1977; Weiter and Pinch, 1975). Our interest in lens fluorescence derives from the interference it produces in measurements of Raman spectra. Both processes involve wavelength Jhifts which are detected by a phototube in the same way. Since Raman emission is generally much weaker than fluorescence, it takes only a low level of fluorescence to bury the Raman scattering in the background fluorescence. Because of this interference no Raman spectra for older human lenses are presently available although new techniques (Ya, 1977) are under development which promise to avoid the interference clue to fluorescence. A young human lens below the age at which fluorescence becomes apparent (about 20 years) does given a Raman spectrum (Pig. 1) which, however; is not different from spectra for other mammalian lenses (Yu and Kuck, 1978).
I 2720
I h 2520 '
I DO0
I 1500
I 500
cm-1
FIG. 1. Raman spectrum of a 6 months old human lens with 511.3 nm excitation. The laser power at the sample is -120 mW and the spectral slit width -5 cm- I. The 2500-2800 cm-l region of the spectrum was recorded at a sensitivity 3 times greater than that used for the 300-1500 cm-l region. The spectra obtained from nucleus and cortex are identical for such a young lens.
Since the two prominent fluorescent materials (called fluorophors by Udenfriend (1962) and others*) in the human lens have well-established wavelength maxima for excitation/emission, 2901340 nm and 340/420nm, we attempted to avoid these * Following the usage of Udenfriend (1962) we term the fluorescent material a fluorophor. This is analogous to the term chromophor applied to a colored molecule. The term fluorogen sometimes used (Lerman and Hollander, 1971) is not in accord with this usage and should be resewed for a nonfluorescent precursor of a fluorophor. 0014-l83~/i8/120737~0~
0 1978 Academic Press Inc. (London) Limited
$01.00/O 737
738
JOHN
P.R.
KUCK,
JR. AND
P;dl-TEXG
YU
exciting wavelengths so as to minimize fluorescence and yet elicit a useful Raman signal. A YS-year-old human lens irradiated with the 600-Onm line of a Chromatix CNX-4 xenon flashlamp-pumped pulsed dye laser gives a satisfactory Raman spectrum (Fig. 2). It appeared possible that using wavelengths farther into the red from the 600.0 nm line might avoid interference from the increased fluorescence of even older lenses.
2580
2731
J$ 2700 2800
2600
NL&US
2500
cm-’ FIG. 2. Kaman spectrum of a 23-year-old human lens (nucleus) with 600.0 nm excitation. The laser power at the sample is -100 mW and the spectral slit width 45 cm-l. The pulsed laser (1 psec pulse width) was operated at 30 Hz. The photomultiplier signal was detected by a Molectron LP-20 boxcar integrator. The -SH intensity at 2580 cm-l (relative to the inkrnal standard at 2731 m-1) is nearly the same as t,hat of the 6 mont,hs old human lens shown in Fig. 1. The 2500-2800 cm-’ region was recorded at ~5 times the amplification of the 300-1800 cm-l region.
In the course of searching for a suitable exciting wavelength we discovered a new fluorophor which is excited maximally at. 514.5 nm and has a broad emission peak with a maximum of 556.4 nm (Fig. 3). This fluorophor is obviously different from the one found by Lerman (1976) with maximum at 420-35 nm and from the P-carbolines, bityrosine and anthranilic acid found by several groups (Dillon, Spector and xakanishi, 1976; Garcia-Castineiras, Dillon and. Spector, 19’78; and Truscot,t, Paul1 and Augusteyn: 1977). Like the one with an emission peak at 420 nm, the new fluorophor increases in concentra.tion with age. It is entirely absent in lenses 7 years of age or younger, it is detectable at 23 years, it makes a significant contribution at 55 years and in the U-year-old lens (Fig. 3) it is still more concentrated. It probably has not been detected before because of its weak emission. We believe our success is due to the use of a Raman spectrometer (Ya, 197.i) as a fluorometer. This instrument may, without modification, serve as a fluorometer with a sensitivity some 100 times that of a conventional fluorometer employing an uncooled photomultiplier tube. Furthermore, because of the small beam waist of the la,ser (Yu, East, Chang and Kuck, 19’77), the instrument may be used to determine exactly the location and concentration of fluorophors in different parts of the lens and even in different parts
LETTER
TO THE
739
EDITORS
of the nucleus, an accomplishment beyond the capability of a fluorometer employing a xenon arc light source. The discovery and indentification of fluorophors in the human lens is of interest because they are possibly key compounds in the formation of high molecular weight protein aggregates found in the nucleus of human nuclear cataracts. !l!he involvement of tryptophan has been discussed by many investigators (Pirie, 1972; Buckingham, 1972; van Heyningen, 1973; Satoh, Bando and Nakajima, 1973; Kurzel, Wolbarsht; Yamanashi, Staton and Borkman, 1973; Weiter and Finch, 1975; Augusteyn, 1975; Lerman, 1976; Truscott and Augusteyn, 1977). Another fluorophor possibly involved
J
- ep.-.--I 3600
2700
1800
__-.-
A__--
J
900
cm-’ FIG. 3. Fluorescent spectra of a W-year-old human lens (nucleus) showing emission maxima at 527-I nm and 5564 nm when the exciting wavelengths are 457.9 nm and 514.5 nm, respectively. The spectra were obtained with the same spectrometer used in Raman experiments.
in aggregation is bityrosine (Garcia-Castineiras, Dillon and Spector, 1978). Such modes of polymerization are in addition to the mechanism postulated many years ago: the 2SH + S-S conversion between two protein molecules, a reaction recently reviewed by Anderson and Spector (1978). Another suggestion is that of Stevens, Rouzer: Monnier and Cerami (1978) that the condensation of glucose with the lysine of crvstallim can give rise to fluorescent products of the Maillard reaction. ;Such a, possfbility recalls the work of Klang (1948) in which he concluded that there was good correlation between the intensity of lens fluorescence and the incidence of cataract in human diabetics. This and subsequent work by Klang and others is reviewed by Kuck (1970). ACKNOWLEDGMEXTS
This work W&Ssupported by Grants EY 00260, EY 01.746 ctnd GXI 188Y4 from the National Institutes of Health. One of us (N.-T. Yu) is a recipient of a National Institutes
740
JOHN
F. R. KUCK,
JR. AND
NAI-TENG
of Health Research Career Development L4ward EY 000'73. Georgia Lions Eye Bank-Atlanta for donor eyes.
“Laboratory for Ophthalwic Research, Emory
Univecsity,
Y L’
We are indebted
JOHNI?.
R. KUCK,
JR.*
NAI-TENG
to the
AXD vut
Atlanta, Ga 30322, U.S.A. $%hool of Chemistry Geoeorgia Institute of Technology, Atlan.ta, Ga 30332, U.S.A. (Received 31 July 1978, New York) REFERENCES Anderson, E. I. and Spector, A. (1978). The state of sulfhydryl groups in normal and cataractous human lens proteins. I. Nuclear region. Ex~. Eye Res. 26, 407-17. Augusteyn, R. (1975). Distribution of fluorescence in the human cataractous lens, O@hnZ. R&s. 7, 217-24. Buckingham, R. H. (1972). The behaviour of reduced proteins from normal and cataractous lenses in high dissociating media: Cross-linked protein in cataractous lenses, Exp. Eye Res. 14,123-Q. Dillon, J., Spector, A. and Nakanishi, Ii. (1976). Identification of P-carbolines isolated from fluorescent human lens proteins, Nature, London 259, 422-3, Garcia-Castineiras, S., Dillon, J. and Spector, 8. (1978). Non-tryptophan fluorescence associated with human lens protein; apparent complexity and isolation of bityrosine and anthranilic acid, Exp. Eye Rea. 26,461-76. Klang, G. (1945). Measurements of the fluorescence of the human lens in vivo, Bctn O$ithnZmoZ. suppz. 31, l-152. Kuck, J. I?. R. (1976). In Biochemistry of tke Eve (C. Graymore, Ed.). P. 221. Academic Press, New York, London. Kurzel, R., Wolbarsht, M. L., Yamanashi, B. S., Smton, G. W. and Borkman, R. F. (1973). Tryptophan excited states and cataracts in the human lens, Nuture, Lond. 241, 132-3. Lerman, S., Louis, D. and Hollander, M. (1971). Characterization of a fluorogen in the ocular lens, Cnn. J. Ophthalmol. 6, 14862. Lerman, S. (1976). Lens fluorescence in aging and cataract formation, DOG. Ophthalmol. 8,241-60. Pirie, A. (1968). Color and solubility of the proteins of human cataract, Inwest. Opkihnlmol. 7, 634-50. Pirie, A. (1972). Photo-oxidation of proteins and compa,rison of phot,o-oxidized protein with those of the cataractous human lens. I.srael. J. illed. Sci. 8, 1567-73. Satoh, Ii., Bando, M. and Nakajima, A. (1973). Fluorescence in human lens, Zqn. @-/e Res. 16, 167-72. Spector, A., Roy, D. and Stauffer, J. (1975). Isolation and characterization of an age-dependent polypeptide from human lens with non-tryptophan fluorescence, Fxp. Eye Res. 21,9-24. Stevens, V. J., R#onzer, C. A., Monnier, 17. M. and Cerami, A. (1978). Diabetic cataract formation: Potential role of glycosylation of lens crystallins. Proc. Nat. Acad. Xci. V.X.A. 75, 2918-22. Truscott, R. J. W. and Augusteyn, R. C. (1977). Changes in human lens proteins during nuclear cataract formation, Exp. Eye Res. 24, 159-70. Truscott, R. J. W., Faull, K. and Augusteyn, R. C. (1977). The identification of anthranilic acid in proteolytie digests of cataractous lens prot,eins. Ophthnl. Res. 9, 263-8. Udenfriend, S. (1962). Proteins. In Fluorescence Assay in Biology and Medicine. Vol. 1, p. 33. Academic Press, New York, London. van Hoyningen, R.. (1973). The glucoside of 3-hydroxykynurenine and other fluorescent compounds in the human Iens. In The Human Lens in Relation to Cntaruct, Ciba Foundation Symposium 19, pp. 151-71. Weiter, J. J. and Finch, E. D. (1975). Paramagnetic species in cataractous human lenses. Nature, London 254,536-7.
LETTER
TO THE
EDITORS
741
Yu, N.-T. (1977). Raman spectroscopy: A conformational probe in biochemistry. CRC Critical Reviews in Biochemistry 4, 229-80. Yu, N.-T., East, E., Chang, R. C. C. and Kuck, J. F. R. (1977). Raman spectra of bird and reptile lens proteins, Exp. Eye Res. 24, 321-34. Yu, X.-T. and Kuck, J. F. R. (1978). F ocusing on lenses with laser Raman spectroscopy. The Spex Speaker. Spex Industries, Metuchen, N.J. (in press) (23:rd year, issue 3). Zigman, 8. (1971). Eye lens color formation and function. Science 171, 807-9.
LETTER
TO THE EDITORS
Raman and Fluorescent Emission of the Human Lens. A New Fluorophor The fluorescence of the human lens is a subject of widespread current interest because of its occurrence at levels elevated much above normal in nuclear senile cataracts (Augnsteyn, 1975; Lerman, Louis and Hollander, 1971; Pirie, 1968; Satoh, Bando and Nakajima, 1973; Spector, Roy and Stauffer; 1975; van Heyningen, 1973; Zigman, 1971). It has been proposed that the accelerated accumula,tion of fluorescent pigment is a manifestation of a polymerization which is part of the same reaction producing the pigment (Buckingham, 1952; Pirie, 1972; Truscott and Augusteyn: 1977; Weiter and Pinch, 1975). Our interest in lens fluorescence derives from the interference it produces in measurements of Raman spectra. Both processes involve wavelength Jhifts which are detected by a phototube in the same way. Since Raman emission is generally much weaker than fluorescence, it takes only a low level of fluorescence to bury the Raman scattering in the background fluorescence. Because of this interference no Raman spectra for older human lenses are presently available although new techniques (Ya, 1977) are under development which promise to avoid the interference clue to fluorescence. A young human lens below the age at which fluorescence becomes apparent (about 20 years) does given a Raman spectrum (Pig. 1) which, however; is not different from spectra for other mammalian lenses (Yu and Kuck, 1978).
I 2720
I h 2520 '
I DO0
I 1500
I 500
cm-1
FIG. 1. Raman spectrum of a 6 months old human lens with 511.3 nm excitation. The laser power at the sample is -120 mW and the spectral slit width -5 cm- I. The 2500-2800 cm-l region of the spectrum was recorded at a sensitivity 3 times greater than that used for the 300-1500 cm-l region. The spectra obtained from nucleus and cortex are identical for such a young lens.
Since the two prominent fluorescent materials (called fluorophors by Udenfriend (1962) and others*) in the human lens have well-established wavelength maxima for excitation/emission, 2901340 nm and 340/420nm, we attempted to avoid these * Following the usage of Udenfriend (1962) we term the fluorescent material a fluorophor. This is analogous to the term chromophor applied to a colored molecule. The term fluorogen sometimes used (Lerman and Hollander, 1971) is not in accord with this usage and should be resewed for a nonfluorescent precursor of a fluorophor. 0014-l83~/i8/120737~0~
0 1978 Academic Press Inc. (London) Limited
$01.00/O 737
738
JOHN
P.R.
KUCK,
JR. AND
P;dl-TEXG
YU
exciting wavelengths so as to minimize fluorescence and yet elicit a useful Raman signal. A YS-year-old human lens irradiated with the 600-Onm line of a Chromatix CNX-4 xenon flashlamp-pumped pulsed dye laser gives a satisfactory Raman spectrum (Fig. 2). It appeared possible that using wavelengths farther into the red from the 600.0 nm line might avoid interference from the increased fluorescence of even older lenses.
2580
2731
J$ 2700 2800
2600
NL&US
2500
cm-’ FIG. 2. Kaman spectrum of a 23-year-old human lens (nucleus) with 600.0 nm excitation. The laser power at the sample is -100 mW and the spectral slit width 45 cm-l. The pulsed laser (1 psec pulse width) was operated at 30 Hz. The photomultiplier signal was detected by a Molectron LP-20 boxcar integrator. The -SH intensity at 2580 cm-l (relative to the inkrnal standard at 2731 m-1) is nearly the same as t,hat of the 6 mont,hs old human lens shown in Fig. 1. The 2500-2800 cm-’ region was recorded at ~5 times the amplification of the 300-1800 cm-l region.
In the course of searching for a suitable exciting wavelength we discovered a new fluorophor which is excited maximally at. 514.5 nm and has a broad emission peak with a maximum of 556.4 nm (Fig. 3). This fluorophor is obviously different from the one found by Lerman (1976) with maximum at 420-35 nm and from the P-carbolines, bityrosine and anthranilic acid found by several groups (Dillon, Spector and xakanishi, 1976; Garcia-Castineiras, Dillon and. Spector, 19’78; and Truscot,t, Paul1 and Augusteyn: 1977). Like the one with an emission peak at 420 nm, the new fluorophor increases in concentra.tion with age. It is entirely absent in lenses 7 years of age or younger, it is detectable at 23 years, it makes a significant contribution at 55 years and in the U-year-old lens (Fig. 3) it is still more concentrated. It probably has not been detected before because of its weak emission. We believe our success is due to the use of a Raman spectrometer (Ya, 197.i) as a fluorometer. This instrument may, without modification, serve as a fluorometer with a sensitivity some 100 times that of a conventional fluorometer employing an uncooled photomultiplier tube. Furthermore, because of the small beam waist of the la,ser (Yu, East, Chang and Kuck, 19’77), the instrument may be used to determine exactly the location and concentration of fluorophors in different parts of the lens and even in different parts
LETTER
TO THE
739
EDITORS
of the nucleus, an accomplishment beyond the capability of a fluorometer employing a xenon arc light source. The discovery and indentification of fluorophors in the human lens is of interest because they are possibly key compounds in the formation of high molecular weight protein aggregates found in the nucleus of human nuclear cataracts. !l!he involvement of tryptophan has been discussed by many investigators (Pirie, 1972; Buckingham, 1972; van Heyningen, 1973; Satoh, Bando and Nakajima, 1973; Kurzel, Wolbarsht; Yamanashi, Staton and Borkman, 1973; Weiter and Finch, 1975; Augusteyn, 1975; Lerman, 1976; Truscott and Augusteyn, 1977). Another fluorophor possibly involved
J
- ep.-.--I 3600
2700
1800
__-.-
A__--
J
900
cm-’ FIG. 3. Fluorescent spectra of a W-year-old human lens (nucleus) showing emission maxima at 527-I nm and 5564 nm when the exciting wavelengths are 457.9 nm and 514.5 nm, respectively. The spectra were obtained with the same spectrometer used in Raman experiments.
in aggregation is bityrosine (Garcia-Castineiras, Dillon and Spector, 1978). Such modes of polymerization are in addition to the mechanism postulated many years ago: the 2SH + S-S conversion between two protein molecules, a reaction recently reviewed by Anderson and Spector (1978). Another suggestion is that of Stevens, Rouzer: Monnier and Cerami (1978) that the condensation of glucose with the lysine of crvstallim can give rise to fluorescent products of the Maillard reaction. ;Such a, possfbility recalls the work of Klang (1948) in which he concluded that there was good correlation between the intensity of lens fluorescence and the incidence of cataract in human diabetics. This and subsequent work by Klang and others is reviewed by Kuck (1970). ACKNOWLEDGMEXTS
This work W&Ssupported by Grants EY 00260, EY 01.746 ctnd GXI 188Y4 from the National Institutes of Health. One of us (N.-T. Yu) is a recipient of a National Institutes
740
JOHN
F. R. KUCK,
JR. AND
NAI-TENG
of Health Research Career Development L4ward EY 000'73. Georgia Lions Eye Bank-Atlanta for donor eyes.
“Laboratory for Ophthalwic Research, Emory
Univecsity,
Y L’
We are indebted
JOHNI?.
R. KUCK,
JR.*
NAI-TENG
to the
AXD vut
Atlanta, Ga 30322, U.S.A. $%hool of Chemistry Geoeorgia Institute of Technology, Atlan.ta, Ga 30332, U.S.A. (Received 31 July 1978, New York) REFERENCES Anderson, E. I. and Spector, A. (1978). The state of sulfhydryl groups in normal and cataractous human lens proteins. I. Nuclear region. Ex~. Eye Res. 26, 407-17. Augusteyn, R. (1975). Distribution of fluorescence in the human cataractous lens, O@hnZ. R&s. 7, 217-24. Buckingham, R. H. (1972). The behaviour of reduced proteins from normal and cataractous lenses in high dissociating media: Cross-linked protein in cataractous lenses, Exp. Eye Res. 14,123-Q. Dillon, J., Spector, A. and Nakanishi, Ii. (1976). Identification of P-carbolines isolated from fluorescent human lens proteins, Nature, London 259, 422-3, Garcia-Castineiras, S., Dillon, J. and Spector, 8. (1978). Non-tryptophan fluorescence associated with human lens protein; apparent complexity and isolation of bityrosine and anthranilic acid, Exp. Eye Rea. 26,461-76. Klang, G. (1945). Measurements of the fluorescence of the human lens in vivo, Bctn O$ithnZmoZ. suppz. 31, l-152. Kuck, J. I?. R. (1976). In Biochemistry of tke Eve (C. Graymore, Ed.). P. 221. Academic Press, New York, London. Kurzel, R., Wolbarsht, M. L., Yamanashi, B. S., Smton, G. W. and Borkman, R. F. (1973). Tryptophan excited states and cataracts in the human lens, Nuture, Lond. 241, 132-3. Lerman, S., Louis, D. and Hollander, M. (1971). Characterization of a fluorogen in the ocular lens, Cnn. J. Ophthalmol. 6, 14862. Lerman, S. (1976). Lens fluorescence in aging and cataract formation, DOG. Ophthalmol. 8,241-60. Pirie, A. (1968). Color and solubility of the proteins of human cataract, Inwest. Opkihnlmol. 7, 634-50. Pirie, A. (1972). Photo-oxidation of proteins and compa,rison of phot,o-oxidized protein with those of the cataractous human lens. I.srael. J. illed. Sci. 8, 1567-73. Satoh, Ii., Bando, M. and Nakajima, A. (1973). Fluorescence in human lens, Zqn. @-/e Res. 16, 167-72. Spector, A., Roy, D. and Stauffer, J. (1975). Isolation and characterization of an age-dependent polypeptide from human lens with non-tryptophan fluorescence, Fxp. Eye Res. 21,9-24. Stevens, V. J., R#onzer, C. A., Monnier, 17. M. and Cerami, A. (1978). Diabetic cataract formation: Potential role of glycosylation of lens crystallins. Proc. Nat. Acad. Xci. V.X.A. 75, 2918-22. Truscott, R. J. W. and Augusteyn, R. C. (1977). Changes in human lens proteins during nuclear cataract formation, Exp. Eye Res. 24, 159-70. Truscott, R. J. W., Faull, K. and Augusteyn, R. C. (1977). The identification of anthranilic acid in proteolytie digests of cataractous lens prot,eins. Ophthnl. Res. 9, 263-8. Udenfriend, S. (1962). Proteins. In Fluorescence Assay in Biology and Medicine. Vol. 1, p. 33. Academic Press, New York, London. van Hoyningen, R.. (1973). The glucoside of 3-hydroxykynurenine and other fluorescent compounds in the human Iens. In The Human Lens in Relation to Cntaruct, Ciba Foundation Symposium 19, pp. 151-71. Weiter, J. J. and Finch, E. D. (1975). Paramagnetic species in cataractous human lenses. Nature, London 254,536-7.
LETTER
TO THE
EDITORS
741
Yu, N.-T. (1977). Raman spectroscopy: A conformational probe in biochemistry. CRC Critical Reviews in Biochemistry 4, 229-80. Yu, N.-T., East, E., Chang, R. C. C. and Kuck, J. F. R. (1977). Raman spectra of bird and reptile lens proteins, Exp. Eye Res. 24, 321-34. Yu, X.-T. and Kuck, J. F. R. (1978). F ocusing on lenses with laser Raman spectroscopy. The Spex Speaker. Spex Industries, Metuchen, N.J. (in press) (23:rd year, issue 3). Zigman, 8. (1971). Eye lens color formation and function. Science 171, 807-9.