- Email: [email protected]
The effect of radiation on collagen I. Electron-spin resonance spectra of 2537-Å-irradiated collagen
222
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 4 5 3 2 4
T H E E F F E C T OF RADIATION ON COLLAGEN I. ELECTRON-SPIN RESONANCE SPECTRA OF 2537-A-IRRADIATED COLLAGEN \V. F. F O R B E S
AND P. D. S U L L [ V A N
Department of Chemistry, University of Waterloo, Waterloo, Ontario (Canada) and Department of Biochemistry, University of Rochester, Rochester, N.Y. (U.S.A.) ( R e c e i v e d O c t o b e r Ist, 1965)
SUMMARY
I. Samples of human collagen, when irradiated with 2537-~ light, afford electron-spin resonance spectra which differ markedly from the electron-spin resonance spectra reported after exposure of collagen samples to X-rays. The signals from ultraviolet light-irradiated collagen are tentatively associated with unpaired electrons located predominantly on the aromatic nuclei of the tyrosine and phenylalauine residues. 2. The collagen signals show characteristic decay rates which also suggest the existence of two distinct radical sites. The decay rates are different for collagen, gelatin and molecular mixtures of approx, the same amino acid composition. 3. The intensities and decay rates of the signals show changes which can be related to the age of the individual samples. 4. On standing, a new electron-spin resonance signal was formed in several cases, which m a y be due to a trapped •CHO radical.
INTRODUCTION
As part of a study to establish on a molecular level the relationship, if any, between the effects of radiation and aging on proteins, the effects of 2537-A light on collagen samples of different age groups have been investigated. Collagen is of obvious interest in aging studies because a considerable proportion of all body proteins is collagen, and there is only a slow cell turnover in collagen, and because the formation of additional cross-linkages in collagen has frequently been postulated as occurring on aging. EXPERIMENTAL
Materials
Collagen samples, obtained from Achilles tendons of males and females aged 0-20 years, 21-3o years and 81-9o years, purified by the method of LABELLA AND Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF 2537-~-IRRADIATED COLLAGEN
223
PAUL1 were used. Less purified collagen afforded qualitatively similar changes to those reported in this paper. Differences may be caused by the known presence of soluble collagen and/or lipids in the latter samples. An amino acid analysis of the 21-3o years and 8I-9 ° years samples gave values for constituent amino acids consistent with the literature s. No significant differences were noted between the two samples. The collagen samples, provided by Dr. LaBELLA, were also treated with 0.2 M acetic acid on a CM-cellulose column and were found to contain only small amounts of soluble collagen; by far the greatest fraction was insoluble collagen. Commercially available amino acids and gelatin (Pfanstiehl Labs. Lot No. 5039) were used.
Irradiations The samples were placed in quartz tubes of external diameter, 4 mm. For irradiations in vacuo, the samples were evacuated and flushed with Nl gas a minimum of four times before sealing at o.I mm Hg. Irradiations were carried out with a 30 W General Electric low pressure germicidal mercury lamp, the output of approx. 4 W consisting mainly of 2537-A light The samples were placed about 3 cm from the light source.
ESR-Spectra ESR spectra were obtained at a microwave frequency of 9ooo Mcycles/sec (X-band) using a J E O L 3BX resonance spectrometer. The traces show the first derivative of the absorption spectrum. The maximum sensitivity of the instrument at room temperature was such that lO11 molecules of diphenylpicrylhydrazyl could readily be detected at room temperature. The magnetic field was calibrated and a "g" marker obtained by simultaneously using a solution of peroxylamine disulphonate in dilute NaHCOa with the sample under investigation. The parameters used for peroxylamine disulphonate were g ---- 2.0o55, total width -~ 26.15 gauss (cf. ref. 3)The magnetic field calibration was also checked against an NMR probe. The relative intensities were calculated as follows: intensity (relative units) is proportional to (height of line). (half width) ~- (factor for gain of amplifier)/(weight of sample). The day to day accuracy of the relative intensities was normally better than 4-5%. Absolute intensities were calculated by comparing signal intensities with signal intensities produced by known concentrations of diphenylpicrylhydrazyl in ZnO. The accuracy of absolute intensities thus determined is estimated to be _-k2o%. RESULTS AND DISCUSSION
Spectra of collagen irradiated by 2537-2~ light Irradiation of the collagen samples afforded an ESR spectrum, as shown in Fig. i. This spectrum is markedly different from that reported by PATTEN AND GORDY4 for y-irradiated collagen, since the latter affords a doublet ESR signal. g-values, intensities and line widths are shown in Table I. Table I shows that the ESR signals, obtained from collagen in vacuo and air, most closely resemble the ESR signals obtained from the irradiated amino acids tyrosine and phenylalanine with respect to line widths and intensities. This would not be unexpected since these two amino acids are able to absorb 2537-A light. Biochim. Biophys. Acta, 12o (1966) 222-228
224
W. F. FORBES, P. D. SULLIVAN
Irradiation of the other amino acids, including tryptophan, with 2537-~ light affords either a much wider signal of approximately the same overall intensity (e.g. leucine, lysine and valine) or, even on prolonged irradiation, much weaker signals of similar overall width (e.g. tryptophan, histidine, serine, elc.) 5. /--- + 19 GAUSS
/~+
a
I0 GAUSS
b
Fig. I. Typical E S R s p e c t r a obtained, at r o o m t e n l p e r a t u r e , in vacuo (a) a n d in air (b), on irradia t i o n w i t h 2 5 3 7 - ~ light, from p o w d e r e d s a m p l e s of collagen (sample from age group 8 1 - 9 o years; d a t a in Fig. i b were o b t a i n e d w i t h t h e amplifier gain increased b y a factor of 4).
Decay rates The growth and decay rates of the ESR signals for various samples of collagen, and for some reference compounds, are shown in Figs. 2 and 3. Fig. 2 shows that on irradiation radical sites are formed more quickly initially, saturation being reached in approx. 2oo h. The decay curves, obtained in the presence of air (Fig. 3; dotted lines), show that the presence of air affects some of the radical sites. The decay rate does not follow a simple exponential curve, indicating that more than one type of radical site is involved. Moreover, since a sample irradiated in air affords a similar signal as the sample irradiated in vacuum but allowed to decay in air (see Table II), it seems likely that the air-sensitive radical gives rise to the initial portion of the decay curves in the presence of air. The presence of more than one radical site is also indicated by the variation of g-values with microwave power (see Table I). Further, the line width data shown in Table II are consistent with the presence of two types of radicals. That is, a signal of line width of about 2o gauss, in the presence of air, changes to a signal of line width of about IO gauss. Since the line widths of the ESR spectra of 2537-A irradiated samples of tyrosine and phenylalanine are about 2o and 13 gauss respectively (see Table I), it is tempting to associate the air-sensitive radical with an unpaired electron located predominantly on the phenylalanine residue, and the less sensitive radical with an unpaired electron located predominantly on the tyrosine residue. It was also found that the ESR spectra of ultraviolet lightirradiated phenylalanine can be explained as the sum of two radical species, one of which has a free electron associated with the side chain 5. The corresponding tyrosine spectrum is interpreted as being due to a single species in which the free electron is Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF 2537-A-IR.RADIATED COLLAGEN
TABLE ESR
225
I DATA FOR COLLAGEN SAMPLES A N D REFERENCE C O M P O U N D S
Reference compounds, De-alanine, L-threonine, e-histidine,L-tryptophan, L-glutanic acid, glycine, L-aspartic acid, L-serine, e-methionine, gave either no signals at all or else signals with an intensity smaller than 4.4" lO17 spins per g. Samples were irradiated in vacuo unless otherwise stated. "g"values were calculated under similar conditions of microwave power, and therefore only hold for these conditions, which were chosen to avoid saturation of the E S R signal. For the collagen in vacuo samples only, a variation of 4- o.ooo4 with microwave power was detected; this, however, m a y be indicative of the two radical species. There was evidence that the collagen signals saturate ; this m a y lead to errors of approx. ~: i.o gauss in the line width. The intensity applies to the m a x i m u m achieved under standard conditions of irradiation.
Compound
"'g"-value
Line width (gauss)
Intensity (spins p e r g × xo 17)
Collagen from age group 0-20 years Collagen from age group 21-3o years Collagen from age group 81-9o years Collagen from age group 0-20 years, irradiated in air Collagen from age group 81-9o years, irradiated in air Gelatin L-Phenylalanine DL-Tyrosine L-Tyrosine Glycyltyrosine DL-Alanylphenylalanine Glycylphenylalanine L-Isoleucine
2.0056 5:o.ooo2 2.0057 5:0.0002 2.0052 4- 0.0002
18.8 -- 0.5 17.9 -¢- 1. 5 20.5 ± 2-5
32 21 42
2.oo5z 4- o.oooi
lO.6
2.0052 2.0053 2.0040 2.0039 2.oo41 2.oo41 2.0035 2.0037 2.OOl 9
L-Valine
2.oo28 -- o.ooo2
L-Leucine
2.0033 -4- o.ooo2
L-Lysine. HC1
2.0032
L-Arginine L-Proline
2.oo41 4- 0.0002 2.0032 5:0.0003
9.6 ± 0.5 21.8 4- 0.8 19.1 2: I.O 14.3 4- 0.3 11.o 7 4- 0. 7 13.4 4- 0.5 24.8 4- I.O 19.9 -¢- 2.0 Complex, overall width about 13o gauss Overall width about I 2 0 gauss Overall width about I O 0 gauss Overall width about i2o gauss 26.1 4- I.O Triplet aH about 17.o gauss
5: o.oooi 5: o.oooi 5:0.0004 5: o.oooi 5:o.ooo2 5:0.0002 ± o.ooo2 5: o.oooi 5: o.oooi
± 0. 3
± 5 ± 3 4- 7
3.0 q- 0. 5 2.5 43 32 8.9 5.0 4.o II.O 14.o 15.o
-¢- 0.3 4- 3 +4 ± 1.4
9.0 12.o 7.0 9.0 8.o
a s s o c i a t e d p r e d o m i n a n t l y w i t h t h e a r o m a t i c r i n g (cf. refs. 6, 7)- T h i s i n d i c a t e s t h a t t h e e l e c t r o n is less f i r m l y h e l d o n t h e a r o m a t i c n u c l e u s of p h e n y l a l a n i n e t h a n of tyrosine'. H o w e v e r , t h e r e a r e difficulties, o r a p p a r e n t difficulties, w i t h s u c h a r a t i o n a l i z a t i o n . N a m e l y , t h e s i g n a l s o b t a i n e d o n 2 5 3 7 - A i r r a d i a t i o n of t y r o s i n e a n d p h e n y l a l a n i n e d o n o t d e c a y i n air. A p o s s i b l e e x p l a n a t i o n of t h i s d i f f i c u l t y is t h a t i n c o l l a g e n t h e free r a d i c a l , a l t h o u g h still p r e d o m i n a n t l y l o c a l i z e d o n t h e a r o m a t i c n u c l e u s , m a y b e a b l e t o m i g r a t e a l o n g t h e h e l i c a l f r a m e w o r k of t h e c o l l a g e n m o l e c u l e . I n t h i s w a y , t h e p a r t l y d e l o c a l i s e d free e l e c t r o n is b o t h m o r e r e a d i l y f o r m e d b u t also a b l e t o r e c o m b i n e m o r e r e a d i l y w i t h a n o t h e r u n p a i r e d e l e c t r o n . T h i s e x p l a n a t i o n c o u l d also r a t i o n a l i z e t h e * Note added in proof: The E S R spectrum of a 2537-A-irradiated sample of poly-L-tyrosine was, in fact, found to be considerably more stable t h a n the corresponding signal from polyL-phenylalanine5. (Received April 7th, 1966) Biochim. Biophys. Acta, 12o (1966) 222-228
226
w.F.
FORBES, P. D. SULLIVAN
6000f\~ " \ x\\
tOi(,
\
0
4000 ~\
1018
\
\\\ \
\-.
\\
"- b
C
?r
1017
0
\ 2000
Z
\
\x
~z I0 t(
iO 15 i
I00
0
I I 200 300 HOURS IRRADIATION
I 400
I 500
I
0
TIME
[
25 HOURS)
50
Fig. 2. G r o w t h r a t e s of t h e E S R signals from (a) collagen in vacuo, (b) gelatin in vaeuo, (c) collagen in air a n d (d) a n a m i n o acid m i x t u r e , of a p p r o x i m a t e l y similar c o m p o s i t i o n as o b t a i n e d in t h e a m i n o acid analysis of collagen, in vacuo. Fig. 3. D e c a y r a t e s for (a) collagen irradiated in vaeuo, (b) gelatin irradiated in vacuo, a n d (c) collagen irradiated in air. F u l l lines show d e c a y rates in absence of air, while d o t t e d lines show d e c a y r a t e s in t h e presence of air. T A B L E II ESR
DATA FOR COLLAGEN SAMPLES IN AIR AND i n v a c u o
Sample
gl
g2
g3
Line width (gauss)
Intensity (relative units)
(i) 81-9o y e a r s
2.OLO6
2.oo51
1.9994
18.4
6900
2.0078
2.0050
2.oo18
9-5
33 °
D e c a y in air for a b o u t 200 h
2.oo78
2.0052
2.oo21
9.65
360
Collagen (duplicate sample) in air (saturated)
2.OllO
2.0054
2.0000
17.8
4400
2.oo81
2.oo51
2.OOl 7
9.8
260
D e c a y in air for a b o u t 20o h
2.0085
2.0052
2.oo21
lO.6
240
Collagen (duplicate sample) in air (saturated)
(2) o - 2 o years
Collagen in vacuo (saturated)
Collagen in vaeuo (saturated)
observations (i) that a mixture of the constituent amino acids afforded a much weaker signal, which, however, did not decay, and (2) that the radical formed in gelatin, which is known not to possess the same regular three-stranded structure as Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF
2537-A-IRRADIATEDCOLLAGEN
227
collagen 8, also decayed more slowly (see Fig. 3 and compare also the relatively greater saturation intensity and faster decay rate in the aged collagen sample--see below).
Age-related changes in the ESR spectra Table I and Fig. 4 show t h a t the older collagen samples afforded a greater "saturation intensity" and also t h a t these samples in v a c u u m initially decayed (logarithmically) more rapidly t h a n the younger collagen samples. Comparable changes are observed for the line-width data. Re-irradiation of a y o u n g collagen sample gave different spectral characteristics 5 but did not afford saturation or decay curves resembling those of an older collagen sample. Consequently, the changes observed on aging are not identical to the changes brought about b y irradiation with 2537-A light. This, of course, does not rule out the possibility t h a t the effects of irradiation p a r t l y contribute to aging b y rupturing some bonds which then slowly recombine to form cross-links.
6000
40OO
2000
LN (TIME IN MINDTES)
6H
Fig. 4. Initial decay rates of the ESR signal for collagen samples from different age groups; (I) 81--9o years, (2) 0-20 years, (3) 21-3o years. Lines are drawn using the method of least squares. Different samples afforded different initial intensities, because of the uncertainty in measuring line widths; however, observed slopes, corresponding to those shown in the figure, were identical within experimental error for different experiments. Fig. 5. Signal obtained from a sample of collagen which had been allowed to stand in air for 2 months.
Changes in the ESR signal on prolonged standing On prolonged standing, certain irradiated amino acids and collagen samples developed a doublet signal showing a splitting of 113-115 gauss and possessing a g-value of 1.9991 -¢- o.oolo (see Fig. 5). The presence of this signal has been detected in some of the following samples: L-tryptophan, L-histidine, L-methionine, L-hydroxyproline, and in various collagens. Possibly, these signals are caused b y a t r a p p e d . CHO radical, formed as a fragmentation product (cf. ref. 9).
Biochim. Biophys. Acta, 12o (1966) 222-228
228
W . F . FORBES, P. D. SULLIVAN
ACKNOWLEDGEMENTS
The authors are indebted to Mr. A. B. BARABAS for competent technical assistance, to Dr. R. R. KOHN for discussions, and to Dr. F. S. LABELLA of the University of Manitoba for generous supplies of purified collagen samples. The research for this paper was supported by the Defence Research Board of Canada, Grant No. 1675-o 4 , the National Research Council of Canada, and by U.S.P.H.S. Grant No. RH oo392-Ol. REFERENCES I F. S. LABELLA AND G. PAUL, J. Gerontol., 20 (1965) 54. 2 J. E. EASTOE, Biochem. J., 61 (1955) 589 • 3 G. VINCOW AND P. M. JOHNSON, J. Chem. Phys., 39 (1963) 1143. 4 R. A. PATTEN AND W. GORDY, Radiation Res., 22 (1964) 29. 5 W. F. FORBES AND P. D. SULLIVAN, unpublished information. 6 R. C. DREW AND W. GORDY, Radiation Res., 18 (1963) 552. 7 0 . A. AZlZOVA, Biofizika, 8 (1964) 556. 8 W. lq'. HARRINGTON AND P. H. VON HIPPEL, Advan. Protein Chem., 16 (1961) I. 9 M. C. R. SY~tONS, in V. GOLD, Advances in Physical Organic Chemistry, Vol. I, Academic Press, New York, 1963, p. 345 and references cited there.
Biochim. Biophys. Acta, 12o (1966) 222-228
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 4 5 3 2 4
T H E E F F E C T OF RADIATION ON COLLAGEN I. ELECTRON-SPIN RESONANCE SPECTRA OF 2537-A-IRRADIATED COLLAGEN \V. F. F O R B E S
AND P. D. S U L L [ V A N
Department of Chemistry, University of Waterloo, Waterloo, Ontario (Canada) and Department of Biochemistry, University of Rochester, Rochester, N.Y. (U.S.A.) ( R e c e i v e d O c t o b e r Ist, 1965)
SUMMARY
I. Samples of human collagen, when irradiated with 2537-~ light, afford electron-spin resonance spectra which differ markedly from the electron-spin resonance spectra reported after exposure of collagen samples to X-rays. The signals from ultraviolet light-irradiated collagen are tentatively associated with unpaired electrons located predominantly on the aromatic nuclei of the tyrosine and phenylalauine residues. 2. The collagen signals show characteristic decay rates which also suggest the existence of two distinct radical sites. The decay rates are different for collagen, gelatin and molecular mixtures of approx, the same amino acid composition. 3. The intensities and decay rates of the signals show changes which can be related to the age of the individual samples. 4. On standing, a new electron-spin resonance signal was formed in several cases, which m a y be due to a trapped •CHO radical.
INTRODUCTION
As part of a study to establish on a molecular level the relationship, if any, between the effects of radiation and aging on proteins, the effects of 2537-A light on collagen samples of different age groups have been investigated. Collagen is of obvious interest in aging studies because a considerable proportion of all body proteins is collagen, and there is only a slow cell turnover in collagen, and because the formation of additional cross-linkages in collagen has frequently been postulated as occurring on aging. EXPERIMENTAL
Materials
Collagen samples, obtained from Achilles tendons of males and females aged 0-20 years, 21-3o years and 81-9o years, purified by the method of LABELLA AND Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF 2537-~-IRRADIATED COLLAGEN
223
PAUL1 were used. Less purified collagen afforded qualitatively similar changes to those reported in this paper. Differences may be caused by the known presence of soluble collagen and/or lipids in the latter samples. An amino acid analysis of the 21-3o years and 8I-9 ° years samples gave values for constituent amino acids consistent with the literature s. No significant differences were noted between the two samples. The collagen samples, provided by Dr. LaBELLA, were also treated with 0.2 M acetic acid on a CM-cellulose column and were found to contain only small amounts of soluble collagen; by far the greatest fraction was insoluble collagen. Commercially available amino acids and gelatin (Pfanstiehl Labs. Lot No. 5039) were used.
Irradiations The samples were placed in quartz tubes of external diameter, 4 mm. For irradiations in vacuo, the samples were evacuated and flushed with Nl gas a minimum of four times before sealing at o.I mm Hg. Irradiations were carried out with a 30 W General Electric low pressure germicidal mercury lamp, the output of approx. 4 W consisting mainly of 2537-A light The samples were placed about 3 cm from the light source.
ESR-Spectra ESR spectra were obtained at a microwave frequency of 9ooo Mcycles/sec (X-band) using a J E O L 3BX resonance spectrometer. The traces show the first derivative of the absorption spectrum. The maximum sensitivity of the instrument at room temperature was such that lO11 molecules of diphenylpicrylhydrazyl could readily be detected at room temperature. The magnetic field was calibrated and a "g" marker obtained by simultaneously using a solution of peroxylamine disulphonate in dilute NaHCOa with the sample under investigation. The parameters used for peroxylamine disulphonate were g ---- 2.0o55, total width -~ 26.15 gauss (cf. ref. 3)The magnetic field calibration was also checked against an NMR probe. The relative intensities were calculated as follows: intensity (relative units) is proportional to (height of line). (half width) ~- (factor for gain of amplifier)/(weight of sample). The day to day accuracy of the relative intensities was normally better than 4-5%. Absolute intensities were calculated by comparing signal intensities with signal intensities produced by known concentrations of diphenylpicrylhydrazyl in ZnO. The accuracy of absolute intensities thus determined is estimated to be _-k2o%. RESULTS AND DISCUSSION
Spectra of collagen irradiated by 2537-2~ light Irradiation of the collagen samples afforded an ESR spectrum, as shown in Fig. i. This spectrum is markedly different from that reported by PATTEN AND GORDY4 for y-irradiated collagen, since the latter affords a doublet ESR signal. g-values, intensities and line widths are shown in Table I. Table I shows that the ESR signals, obtained from collagen in vacuo and air, most closely resemble the ESR signals obtained from the irradiated amino acids tyrosine and phenylalanine with respect to line widths and intensities. This would not be unexpected since these two amino acids are able to absorb 2537-A light. Biochim. Biophys. Acta, 12o (1966) 222-228
224
W. F. FORBES, P. D. SULLIVAN
Irradiation of the other amino acids, including tryptophan, with 2537-~ light affords either a much wider signal of approximately the same overall intensity (e.g. leucine, lysine and valine) or, even on prolonged irradiation, much weaker signals of similar overall width (e.g. tryptophan, histidine, serine, elc.) 5. /--- + 19 GAUSS
/~+
a
I0 GAUSS
b
Fig. I. Typical E S R s p e c t r a obtained, at r o o m t e n l p e r a t u r e , in vacuo (a) a n d in air (b), on irradia t i o n w i t h 2 5 3 7 - ~ light, from p o w d e r e d s a m p l e s of collagen (sample from age group 8 1 - 9 o years; d a t a in Fig. i b were o b t a i n e d w i t h t h e amplifier gain increased b y a factor of 4).
Decay rates The growth and decay rates of the ESR signals for various samples of collagen, and for some reference compounds, are shown in Figs. 2 and 3. Fig. 2 shows that on irradiation radical sites are formed more quickly initially, saturation being reached in approx. 2oo h. The decay curves, obtained in the presence of air (Fig. 3; dotted lines), show that the presence of air affects some of the radical sites. The decay rate does not follow a simple exponential curve, indicating that more than one type of radical site is involved. Moreover, since a sample irradiated in air affords a similar signal as the sample irradiated in vacuum but allowed to decay in air (see Table II), it seems likely that the air-sensitive radical gives rise to the initial portion of the decay curves in the presence of air. The presence of more than one radical site is also indicated by the variation of g-values with microwave power (see Table I). Further, the line width data shown in Table II are consistent with the presence of two types of radicals. That is, a signal of line width of about 2o gauss, in the presence of air, changes to a signal of line width of about IO gauss. Since the line widths of the ESR spectra of 2537-A irradiated samples of tyrosine and phenylalanine are about 2o and 13 gauss respectively (see Table I), it is tempting to associate the air-sensitive radical with an unpaired electron located predominantly on the phenylalanine residue, and the less sensitive radical with an unpaired electron located predominantly on the tyrosine residue. It was also found that the ESR spectra of ultraviolet lightirradiated phenylalanine can be explained as the sum of two radical species, one of which has a free electron associated with the side chain 5. The corresponding tyrosine spectrum is interpreted as being due to a single species in which the free electron is Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF 2537-A-IR.RADIATED COLLAGEN
TABLE ESR
225
I DATA FOR COLLAGEN SAMPLES A N D REFERENCE C O M P O U N D S
Reference compounds, De-alanine, L-threonine, e-histidine,L-tryptophan, L-glutanic acid, glycine, L-aspartic acid, L-serine, e-methionine, gave either no signals at all or else signals with an intensity smaller than 4.4" lO17 spins per g. Samples were irradiated in vacuo unless otherwise stated. "g"values were calculated under similar conditions of microwave power, and therefore only hold for these conditions, which were chosen to avoid saturation of the E S R signal. For the collagen in vacuo samples only, a variation of 4- o.ooo4 with microwave power was detected; this, however, m a y be indicative of the two radical species. There was evidence that the collagen signals saturate ; this m a y lead to errors of approx. ~: i.o gauss in the line width. The intensity applies to the m a x i m u m achieved under standard conditions of irradiation.
Compound
"'g"-value
Line width (gauss)
Intensity (spins p e r g × xo 17)
Collagen from age group 0-20 years Collagen from age group 21-3o years Collagen from age group 81-9o years Collagen from age group 0-20 years, irradiated in air Collagen from age group 81-9o years, irradiated in air Gelatin L-Phenylalanine DL-Tyrosine L-Tyrosine Glycyltyrosine DL-Alanylphenylalanine Glycylphenylalanine L-Isoleucine
2.0056 5:o.ooo2 2.0057 5:0.0002 2.0052 4- 0.0002
18.8 -- 0.5 17.9 -¢- 1. 5 20.5 ± 2-5
32 21 42
2.oo5z 4- o.oooi
lO.6
2.0052 2.0053 2.0040 2.0039 2.oo41 2.oo41 2.0035 2.0037 2.OOl 9
L-Valine
2.oo28 -- o.ooo2
L-Leucine
2.0033 -4- o.ooo2
L-Lysine. HC1
2.0032
L-Arginine L-Proline
2.oo41 4- 0.0002 2.0032 5:0.0003
9.6 ± 0.5 21.8 4- 0.8 19.1 2: I.O 14.3 4- 0.3 11.o 7 4- 0. 7 13.4 4- 0.5 24.8 4- I.O 19.9 -¢- 2.0 Complex, overall width about 13o gauss Overall width about I 2 0 gauss Overall width about I O 0 gauss Overall width about i2o gauss 26.1 4- I.O Triplet aH about 17.o gauss
5: o.oooi 5: o.oooi 5:0.0004 5: o.oooi 5:o.ooo2 5:0.0002 ± o.ooo2 5: o.oooi 5: o.oooi
± 0. 3
± 5 ± 3 4- 7
3.0 q- 0. 5 2.5 43 32 8.9 5.0 4.o II.O 14.o 15.o
-¢- 0.3 4- 3 +4 ± 1.4
9.0 12.o 7.0 9.0 8.o
a s s o c i a t e d p r e d o m i n a n t l y w i t h t h e a r o m a t i c r i n g (cf. refs. 6, 7)- T h i s i n d i c a t e s t h a t t h e e l e c t r o n is less f i r m l y h e l d o n t h e a r o m a t i c n u c l e u s of p h e n y l a l a n i n e t h a n of tyrosine'. H o w e v e r , t h e r e a r e difficulties, o r a p p a r e n t difficulties, w i t h s u c h a r a t i o n a l i z a t i o n . N a m e l y , t h e s i g n a l s o b t a i n e d o n 2 5 3 7 - A i r r a d i a t i o n of t y r o s i n e a n d p h e n y l a l a n i n e d o n o t d e c a y i n air. A p o s s i b l e e x p l a n a t i o n of t h i s d i f f i c u l t y is t h a t i n c o l l a g e n t h e free r a d i c a l , a l t h o u g h still p r e d o m i n a n t l y l o c a l i z e d o n t h e a r o m a t i c n u c l e u s , m a y b e a b l e t o m i g r a t e a l o n g t h e h e l i c a l f r a m e w o r k of t h e c o l l a g e n m o l e c u l e . I n t h i s w a y , t h e p a r t l y d e l o c a l i s e d free e l e c t r o n is b o t h m o r e r e a d i l y f o r m e d b u t also a b l e t o r e c o m b i n e m o r e r e a d i l y w i t h a n o t h e r u n p a i r e d e l e c t r o n . T h i s e x p l a n a t i o n c o u l d also r a t i o n a l i z e t h e * Note added in proof: The E S R spectrum of a 2537-A-irradiated sample of poly-L-tyrosine was, in fact, found to be considerably more stable t h a n the corresponding signal from polyL-phenylalanine5. (Received April 7th, 1966) Biochim. Biophys. Acta, 12o (1966) 222-228
226
w.F.
FORBES, P. D. SULLIVAN
6000f\~ " \ x\\
tOi(,
\
0
4000 ~\
1018
\
\\\ \
\-.
\\
"- b
C
?r
1017
0
\ 2000
Z
\
\x
~z I0 t(
iO 15 i
I00
0
I I 200 300 HOURS IRRADIATION
I 400
I 500
I
0
TIME
[
25 HOURS)
50
Fig. 2. G r o w t h r a t e s of t h e E S R signals from (a) collagen in vacuo, (b) gelatin in vaeuo, (c) collagen in air a n d (d) a n a m i n o acid m i x t u r e , of a p p r o x i m a t e l y similar c o m p o s i t i o n as o b t a i n e d in t h e a m i n o acid analysis of collagen, in vacuo. Fig. 3. D e c a y r a t e s for (a) collagen irradiated in vaeuo, (b) gelatin irradiated in vacuo, a n d (c) collagen irradiated in air. F u l l lines show d e c a y rates in absence of air, while d o t t e d lines show d e c a y r a t e s in t h e presence of air. T A B L E II ESR
DATA FOR COLLAGEN SAMPLES IN AIR AND i n v a c u o
Sample
gl
g2
g3
Line width (gauss)
Intensity (relative units)
(i) 81-9o y e a r s
2.OLO6
2.oo51
1.9994
18.4
6900
2.0078
2.0050
2.oo18
9-5
33 °
D e c a y in air for a b o u t 200 h
2.oo78
2.0052
2.oo21
9.65
360
Collagen (duplicate sample) in air (saturated)
2.OllO
2.0054
2.0000
17.8
4400
2.oo81
2.oo51
2.OOl 7
9.8
260
D e c a y in air for a b o u t 20o h
2.0085
2.0052
2.oo21
lO.6
240
Collagen (duplicate sample) in air (saturated)
(2) o - 2 o years
Collagen in vacuo (saturated)
Collagen in vaeuo (saturated)
observations (i) that a mixture of the constituent amino acids afforded a much weaker signal, which, however, did not decay, and (2) that the radical formed in gelatin, which is known not to possess the same regular three-stranded structure as Biochim. Biophys. Acta, 12o (1966) 222-228
ESR
SPECTRA OF
2537-A-IRRADIATEDCOLLAGEN
227
collagen 8, also decayed more slowly (see Fig. 3 and compare also the relatively greater saturation intensity and faster decay rate in the aged collagen sample--see below).
Age-related changes in the ESR spectra Table I and Fig. 4 show t h a t the older collagen samples afforded a greater "saturation intensity" and also t h a t these samples in v a c u u m initially decayed (logarithmically) more rapidly t h a n the younger collagen samples. Comparable changes are observed for the line-width data. Re-irradiation of a y o u n g collagen sample gave different spectral characteristics 5 but did not afford saturation or decay curves resembling those of an older collagen sample. Consequently, the changes observed on aging are not identical to the changes brought about b y irradiation with 2537-A light. This, of course, does not rule out the possibility t h a t the effects of irradiation p a r t l y contribute to aging b y rupturing some bonds which then slowly recombine to form cross-links.
6000
40OO
2000
LN (TIME IN MINDTES)
6H
Fig. 4. Initial decay rates of the ESR signal for collagen samples from different age groups; (I) 81--9o years, (2) 0-20 years, (3) 21-3o years. Lines are drawn using the method of least squares. Different samples afforded different initial intensities, because of the uncertainty in measuring line widths; however, observed slopes, corresponding to those shown in the figure, were identical within experimental error for different experiments. Fig. 5. Signal obtained from a sample of collagen which had been allowed to stand in air for 2 months.
Changes in the ESR signal on prolonged standing On prolonged standing, certain irradiated amino acids and collagen samples developed a doublet signal showing a splitting of 113-115 gauss and possessing a g-value of 1.9991 -¢- o.oolo (see Fig. 5). The presence of this signal has been detected in some of the following samples: L-tryptophan, L-histidine, L-methionine, L-hydroxyproline, and in various collagens. Possibly, these signals are caused b y a t r a p p e d . CHO radical, formed as a fragmentation product (cf. ref. 9).
Biochim. Biophys. Acta, 12o (1966) 222-228
228
W . F . FORBES, P. D. SULLIVAN
ACKNOWLEDGEMENTS
The authors are indebted to Mr. A. B. BARABAS for competent technical assistance, to Dr. R. R. KOHN for discussions, and to Dr. F. S. LABELLA of the University of Manitoba for generous supplies of purified collagen samples. The research for this paper was supported by the Defence Research Board of Canada, Grant No. 1675-o 4 , the National Research Council of Canada, and by U.S.P.H.S. Grant No. RH oo392-Ol. REFERENCES I F. S. LABELLA AND G. PAUL, J. Gerontol., 20 (1965) 54. 2 J. E. EASTOE, Biochem. J., 61 (1955) 589 • 3 G. VINCOW AND P. M. JOHNSON, J. Chem. Phys., 39 (1963) 1143. 4 R. A. PATTEN AND W. GORDY, Radiation Res., 22 (1964) 29. 5 W. F. FORBES AND P. D. SULLIVAN, unpublished information. 6 R. C. DREW AND W. GORDY, Radiation Res., 18 (1963) 552. 7 0 . A. AZlZOVA, Biofizika, 8 (1964) 556. 8 W. lq'. HARRINGTON AND P. H. VON HIPPEL, Advan. Protein Chem., 16 (1961) I. 9 M. C. R. SY~tONS, in V. GOLD, Advances in Physical Organic Chemistry, Vol. I, Academic Press, New York, 1963, p. 345 and references cited there.
Biochim. Biophys. Acta, 12o (1966) 222-228