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Photochemistry of Amino Acids and Proteins
5 Photochemistry of Amino Acids and Proteins 5-1. 5-2. 5-3. 5-4.
5-1.
Introduction Relative Photochemical Sensitivity of the Amino Acids General Comments on the Photochemistry of Proteins . Photochemical Inactivation of Enzymes General References
85 86 88 91 95
INTRODUCTION
During the past 10-15 years, general interest in photobiology has strongly emphasized the nucleic acids, although currently there is r e n e w e d interest in p h o t o n effects on proteins. M o s t of the d a t a on the photochemical reactions of the amino acids, h o w e v e r , w e r e gathered s o m e years ago without the benefit of m o n o c h r o m a t i c light sources, the availability of labeled amino acids, or of m o d e r n m e t h o d s of c h r o m a t o g r a p h y and electrophoresis for the separation of the p h o t o p r o d u c t s . T h e reported q u a n t u m yields for proteins are sometimes in error due to inadequate knowledge (at that time) of the molecular weight and amino acid composition of the particular proteins u n d e r investigation. T h e ready availability of synthetic polyamino acids now provides an excellent opportunity for further exciting work on the photochemistry of the amino acids especially as regards possible energy migration and neighbor interactions. O u r understanding of the effects of U V o n cells requires an u n d e r s t a n d ing of the chemical modifications p r o d u c e d in proteins by U V . T h e finding that proteins and D N A interact photochemically within the cell (Section 4-9) further necessitates a renaissance in studies on the photochemistry of amino acids and proteins. 85
5.
86
5-2.
RELATIVE
PHOTOCHEMISTRY OF AMINO ACIDS A N D PROTEINS
PHOTOCHEMICAL SENSITIVITY OF THE
AMINO
ACIDS
Since light must be a b s o r b e d before photochemistry will occur, it is reasonable to a s s u m e that those amino acids that are transparent to the wavelengths normally used in photobiology (wavelengths greater than 2 4 0 0 Ä ) will undergo little or no p h o t o c h e m i s t r y at these wavelengths. T h i s then largely eliminates the aliphatic a m i n o acids from our consideration. T h e strong absorption of wavelengths above 2 4 0 0 A by the aromatic amino acids would certainly suggest their photochemical importance, although it should be recalled that absorbed light need hot necessarily result in p h o t o c h e m i s t r y (Sec tion 1 - 2 ) . Before continuing with the photochemistry of the aromatic amino acids, let us consider two c h r o m o p h o r e s that are not present in monomeric amino acids but are present in proteins: peptide linkages and disulfide bridges. T h e absorption spectrum for peptide b o n d s shows a peak in the region of 1 8 0 0 - 1 9 0 0 A which d e c r e a s e s essen tially to zero by about 2 4 0 0 Ä . Peptide b o n d s are b r o k e n by ab sorption at these wavelengths. Therefore, if peptide bond breakage is to be minimized, wavelengths below 2 4 0 0 Ä must be filtered out of the light source. Even at 2 5 3 7 Ä , however, the q u a n t u m yield for peptide bond breakage is a significant value ( T a b l e 5 - 1 ) . T h e amino acids, cysteine and cystine, were considered at one time to be unimportant photochemically since according to their absorption spectra in acid solution they w e r e essentially optically transparent a b o v e 2 3 0 0 A. A t neutral and alkaline p H , h o w e v e r , the absorption by these c o m p o u n d s b e c o m e s appreciable even at wavelengths above 2 5 0 0 Ä . T h i s fact coupled with the high q u a n t u m efficiency for their photochemical alteration m a k e s cystine the most sensitive target affecting protein function. If the molar absorptivity (e) of a c o m p o u n d is a m e a s u r e of the probability that light of a particular wavelength will be absorbed by that c o m p o u n d , and the q u a n t u m yield ( Φ ) is the probability that the absorbed light will cause a chemical change, then the p r o d u c t of these two values (e Χ Φ ) is a m e a s u r e of the photochemical sensi tivity of that c o m p o u n d . Values for these p a r a m e t e r s at 2 5 3 7 A for the most important c h r o m o p h o r e s in proteins are given in T a b l e 5 - 1 . T h u s , w h e n cystine is present in a protein it is the most labile target. Following in decreasing lability are t r y p t o p h a n , phenylalanine,
5-2.
PHOTOCHEMICAL SENSITIVITY OF AMINO ACIDS
TABLE 5-1.
87
T H E RELATIVE PHOTOCHEMICAL LABILITY OF AMINO ACIDS
AT 2537 A« b
0
Compound
e
φο
(e Χ Φ)
Cystine (—S—S— bond) Tryptophan Phenylalanine Tyrosine Peptide bonds (acetylalanine) Histidine
270 2870 140 320 0.2 0.24
0.13 0.004 0.013 0.002 0.05 <0.03
35.1 11.5 1.8 0.6 0.01 <0.0072
"Data from A. D. McLaren and D. Shugar, "Photochemistry of Proteins and Nucleic Acids," p. 97. Pergamon Press, Oxford, 1964. b e indicates molar absorptivity at 2537 A, or the probability that light at 2537 Λ will be absorbed by the given compound. ""Φ indicates quantum yield (the probability that the absorbed light (2537 Ä) will cause a chemical change). d (e x Φ) indicates a measure of the photochemical sensitivity of a particular amino acid (it is related to the inactivation cross-section, cr, by a constant; Section 6-2).
tyrosine, peptide b o n d s , and histidine. It should be stressed, how ever, that at other wavelengths the U V sensitivity and therefore the relative importance of these targets in the photochemical in activation of a particular protein will change. Most of the early photochemical studies on the amino acids did not use m o n o c h r o m a t i c light and the analysis for photochemical alteration relied upon easily measurable p r o d u c t s such as ammonia, carbon dioxide, acids, b a s e s , sulfur and loss of spectral properties. T h e importance of using m o n o c h r o m a t i c light for photochemical studies has already been stressed (e.g., Section 4-5). Relying only upon end products of destruction such as a m m o n i a , c a r b o n dioxide, etc., very little information can be gained about intermediate reac tions that o c c u r at biologically significant d o s e s of U V . A recent study on the p h o t o c h e m i s t r y of phenylalanine (selec 14 tively labeled at different positions with C ) has s h o w n that w h e n this amino acid is irradiated at the wavelength of m a x i m u m a b s o r p tion of the aromatic residue (2575 Ä) that tyrosine, dihydroxyphenylalanine ( D O P A ) , aspartic acid, benzoic acid, phenyllactic acid, and a high molecular weight melanic polymer are formed. T h e reaction that occurred with the greatest q u a n t u m efficiency w a s the liberation of carbon dioxide by decarboxylation. T h e s e results indicate that there w a s cleavage of side chain c a r b o n b o n d s , and
88
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PHOTOCHEMISTRY OF AMINO ACIDS A N D
PROTEINS
since all of the energy was initially absorbed by the b e n z e n e ring, it is therefore reasonable to conclude that energy migration by some mechanism (as yet not understood) must have occurred. T h e direct transmission of energy along — ( C H 2 ) W— would a p p e a r to b e incon sistent with the observation (Section 1-3) that m e t h y l e n e groups affect the absorption spectra of c o m p o u n d s by acting as insulators between aromatic groups. Several other examples of energy migration in peptides and pro teins are known. T h e photochemical decomposition of c a r b o n monoxide-myoglobin o c c u r s with the same q u a n t u m yield regard less of whether the light energy is absorbed by the aromatic amino acids of the protein or the porphyrin ring. W h e n the d y e 1-dimethylaminonaphthalene-5-sulfonyl chloride w a s b o u n d to proteins and the complex irradiated with light that was specifically absorbed by the aromatic residues of the protein it was found that the d y e fluoresced. T h e fluorescence of the d y e could only be explained on the basis of energy transfer from the protein. This type of response has also been shown to result in a d e c r e a s e in the inactivation rate of ir radiated proteins. Energy migration is thought to o c c u r in part through a resonance-transfer mechanism. A fluorescent donor, with a fluorescence spectrum overlapping the absorption spectrum of the energy acceptor, can pass energy to the a c c e p t o r if the dis tance between the two is small (less than 100 Λ) and if the relative orientation b e t w e e n the t w o is suitable (Section 3-7). Since it is possible to distinguish b e t w e e n the spectra of phenylalanine, tyro sine and tryptophan the fluorescence and p h o s p h o r e s c e n c e spectra of proteins can yield information on the direction of transfer of energy b e t w e e n these amino acids. Phenylalanine fluorescence is not observed from most proteins although a tyrosine c o m p o n e n t is always observed. T y r o s i n e fluorescence m a k e s only a small con tribution to the total fluorescence from those proteins containing tryptophan. T h e yields of cystine destruction are m u c h higher in those proteins that contain tryptophan, again emphasizing the im portance of energy transfer in an irradiated protein (Section 5-4). 5-3.
GENERAL
COMMENTS
ON
THE
PHOTOCHEMISTRY
OF
PROTEINS
(a) W h e n proteins are irradiated with U V , both lower and higher molecular weight products are formed. T h e higher molecular weight
5-3.
G E N E R A L C O M M E N T S O N PHOTOCHEMISTRY O F PROTEINS
89
aggregates are m u c h easier to detect, h o w e v e r , and a p p e a r to be produced with the greatest efficiency. While aggregation p r o c e e d s at the same rate u n d e r oxygen or nitrogen at lower d o s e s of U V , at higher d o s e s in the p r e s e n c e of oxygen, the aggregates begin to d e c o m p o s e and the solubility of the irradiated protein therefore begins to increase. (b) Although certain aspects of the p h o t o c h e m i s t r y of proteins are dependent upon w h e t h e r they are irradiated in the p r e s e n c e of oxygen (see above), the q u a n t u m yield for inactivation of an e n z y m e does not d e p e n d upon the a t m o s p h e r e during irradiation. (c) Irradiated proteins are much more susceptible to enzymatic digestion. T h i s , coupled with the fact that heat d e n a t u r e d proteins are in general more susceptible to e n z y m a t i c digestion, indicates that U V c a u s e s the denaturation of proteins. It has also b e e n o b served that following d o s e s of U V (which c a u s e no serious loss in enzymatic activity) the e n z y m e s are m u c h more sensitive to subse quent heat inactivation. At least t w o m e c h a n i s m s could explain this observation. If a peptide bond were b r o k e n but the b r o k e n ends were held in place by the hydrogen b o n d s involved in the sec ondary and tertiary structures of the protein, then the molecule would be enzymatically active until gently heat treated. If a disulfide bridge were broken by U V , then it might require less heat to dis organize the hydrogen bonds of the molecule and in the a b s e n c e of the directional influence of the disulfide bridge, the original hydrogen b o n d s would be less likely to reform on cooling than they would have prior to irradiation. (d) N e a r room t e m p e r a t u r e , the q u a n t u m yield for the inactiva tion of an e n z y m e does not show any m a r k e d d e p e n d e n c y upon the temperature during irradiation. H o w e v e r , at t e m p e r a t u r e s below room t e m p e r a t u r e there is a continuous d e c r e a s e (as the tempera ture decreases) in the q u a n t u m yield; while as the t e m p e r a t u r e in creases a b o v e r o o m t e m p e r a t u r e , there is a c o n t i n u o u s increase in q u a n t u m yield. W e have c o m m e n t e d previously on the effect of temperature and environment upon the a b s o r b a n c e of various com pounds ( C h a p t e r 1) and on the photochemical reactivity of D N A (Chapter 4). Such considerations may possibly be invoked to explain the effect of t e m p e r a t u r e on the photochemical reactivity of proteins. (e) T h e q u a n t u m yields for the U V inactivation of e n z y m e s irradiated in the dry state and in a q u e o u s m e d i u m d o not differ significantly. This suggests that the m e c h a n i s m s involved in the
90
5.
PHOTOCHEMISTRY OF AMINO ACIDS A N D
PROTEINS
two cases are not radically different, but this may well be an over simplification. (f) T h e q u a n t u m yield for proteins varies with the wavelength of irradiation. T h e explanation for this is that the action s p e c t r u m for the inactivation of an e n z y m e can be resolved into the contributions by the various c h r o m o p h o r e s . At 2537 Ä , the q u a n t u m yield for the inactivation of trypsin can be adequately accounted for by assigning the major role to cystine, but at wavelengths a b o v e this value the aromatic amino acids play a more important role and at wavelengths below 2537 A peptide b o n d s play an important role. (g) T h e q u a n t u m yield (at 2537 Ä) for the inactivation of an e n z y m e is roughly proportional to its cystine content. T h i s is con sistent with the data cited in T a b l e 5-1 and will be more fully dis cussed in Section 5-4. (h) T h e q u a n t u m yield for the inactivation of an e n z y m e is roughly T A B L E 5-2.
SOME CHANGES PRODUCED IN PROTEINS FOLLOWING EXPOSURE TO
Property
U V
Change
Color Solubility in water
Darkens Decreases; then increases
Sensitivity to heat
Usually increases
Optical rotation Change in absorption
Increase in levorotation Increases
Digestibility by trypsin Exposure of SH groups Exposure of acid groups
Increases Increases Increase; pH change
Electrophoretic pattern Ultracentrifugation Immunological effects
Heterogenization Increased broadening of sedimentation bands Destruction of antigenic properties; formation of new ones
Possible meaning Photooxidation Aggregation, then degradation Hydrophobic groups exposed; chain breaks Structural changes Depolymerization and photooxidation; formation of new chromophores Opening up of molecule Opening up of molecule Photooxidation to form more phenols; breakage of peptide bonds Degradation and reaggregation Molecular weight hetero geneity Altered amino acid residues
"Modified from A. C. Giese, Physiol. Rev. 30, 431 (1950).
5-4.
PHOTOCHEMICAL I N A C T I V A T I O N O F E N Z Y M E S
91
proportional to the reciprocal of its molecular weight. A precise explanation for this observation is not available, h o w e v e r , it has been pointed out that the cystine content of proteins is roughly proportional to the reciprocal of their molecular weights. (i) In most cases the q u a n t u m yields for the inactivation of the nucleic acids are less than those for proteins, yet the molar a b s o r b tivity for the proteins are m u c h lower t h a n for t h e nucleic acids. M c L a r e n has o b s e r v e d that the p r o d u c t of the molar absorptivity (e) and q u a n t u m yield (Φ) for proteins at 2537 A is approximately equal to that for the nucleic acids. T h e significance of this o b s e r v a tion remains to be evaluated but at face value it would imply that proteins may play a larger role in biological inactivation than most of the nucleic acid chemists would c a r e to admit. (j) T h e q u a n t u m yields for the inactivation of e n z y m e s usually vary with p H but there a p p e a r s to be no correlation with the iso electric point of the e n z y m e . T h e overall charge on the molecule would therefore appear to be of less importance than the charge on some specific amino acid residue(s). (k) O t h e r changes noted in proteins following e x p o s u r e to U V are listed in Table 5-2. 5-4.
PHOTOCHEMICAL
INACTIVATION OF
ENZYMES
T w o different theories of the m e c h a n i s m s involved in the U V inactivation of e n z y m e s h a v e d e v e l o p e d o v e r a period of y e a r s . M c L a r e n and c o - w o r k e r s h a v e a s s u m e d , as a first approximation, that the alteration of any amino acid residue c a u s e s inactivation of an e n z y m e . If the quantity (e x Φ) for each amino acid (which is a measure of the photochemical sensitivity of each amino acid; see T a b l e 5-1) is multiplied by the n u m b e r of such amino acids (n) in the protein and the sum of these is divided by the molar a b sorptivity of the e n z y m e , one arrives at a calculated q u a n t u m yield for the inactivation of the e n z y m e which in s o m e c a s e s is very close to the observed q u a n t u m yield. Of the ten proteins listed in T a b l e 5-3, the calculated q u a n t u m yields for seven of these are within a factor of 2 of the experimentally determined values. T h e values for c a r b o x y p e p t i d a s e , pepsin, and insulin do not a d e q u a t e l y fit the hypothesis. T h e authors themselves h a v e pointed u p certain criticisms of
92
5.
PHOTOCHEMISTRY OF AMINO ACIDS AND PROTEINS
these calculations. T h e r e is no a s s u r a n c e that either the molar absorptivity or the q u a n t u m yield for a particular amino acid (at a given p H and wavelength) is the s a m e in a protein as it is in free solution; in fact, there is s o m e evidence that these values change w h e n the amino acid is incorporated into a protein. Also it is k n o w n that all residues of a particular amino acid in a given protein are not of equal importance to enzymic function. F o r those few proteins studied, their a b s o r b a n c e at 2537 Á d o e s not differ markedly from that for an equivalent mixture of the free amino acids although greater differences exist at other wavelengths. This fact, plus the rather close agreement b e t w e e n the calculated and determined q u a n t u m yields cited in T a b l e 5-3 would suggest that u n d e r certain conditions the amino acid residues are, in fact, independent absorbers unaffected by their neighbors. M c L a r e n and co-workers therefore state that energy transfer a m o n g c h r o m o p h o r e s within molecules of e n z y m e s need not be invoked in o r d e r to account for photochemical inactivation. Augenstein and associates, h o w e v e r , take particular exception to this conclusion. T h e y have developed a theory that e n z y m e s are inactivated by U V as a c o n s e q u e n c e of the disruption of specific cystine residues and of hydrogen b o n d s responsible for the spatial integrity of the active center of the e n z y m e . T h e i r theory e m p h a s i z e s the importance of energy migration in this p r o c e s s . C o n s i s t e n t with these postulates are the observations that the photochemical d a m a g e in the e n z y m e ribonuclease is n o n r a n d o m ; at least t w o and p e r h a p s three of the four constituent cystines must b e disrupted before activity is lost, i.e., the most photosensitive cystines are not critical for enzymic activity. Similarly, in both trypsin and l y s o z y m e the integrity of the m o s t photosensitive cystines d o e s not a p p e a r to b e critical for the retention of enzymic potential. In insulin, h o w e v e r , all three cystines a p p e a r to be crucial for activity and h a v e approxi mately equal photosensitivities. T h e s e differences in sensitivity of cystines in different proteins must d e p e n d specifically u p o n energy transfer a n d / o r chemical interactions b e t w e e n the c h r o m o p h o r i c groups. If q u a n t u m yields are calculated on the basis of those q u a n t a absorbed only in the cystines, values for cystine destruction in proteins are obtained which are a b o u t 5 to 8 times greater than those observed in the model c o m p o u n d s , cystine and oxidized glutathione. Cystine destruction is m u c h greater in those proteins which contain
5-4.
93
PHOTOCHEMICAL INACTIVATION OF ENZYMES
tryptophan. T h e r e is s o m e evidence that the most photosensitive cystines lie close to t r y p t o p h a n residues as a c o n s e q u e n c e of the tertiary structure of the e n z y m e rather than of its primary structure. W e seem to be faced with t w o opposing theories c o n c e r n i n g the involvement of energy transfer in the U V inactivation of e n z y m e s .
T A B L E 5-3.
CALCULATED AND K N O W N QUANTUM YIELDS FOR ENZYME
INACTIVATION BY UV (2537 A)°
Quantum yield Protein
Chromophores
Found
1. Carboxypeptidase 2. Chymotrypsin
Cys 2- His 8- Phe 15 •Try 6 • Tyr 20 Cys 5- His 2 · Phe 6- Try 7 · Tyr 4
0.00 l 0.005
3. Lysozyme
Cys 5- His!- Phe 3- Try 8 · Tyr 2
0.024
4. Pepsin
Cys 3- His 2 · Phe 9- Try 4 · Tyr 16
0.002
5. Ribonuclease
Cys 4- His 4 · Phe 3- Try 0 · Tyr 16
0.027
6. 7. 8. 9.
Cys 0CysoCysoCys 6-
Subtilisin A Subtilisin Β Japanese Nagarse Trypsin
10. Insulin
His 5His 6His 6His x-
Phe 4Phe 3Phe 3Phe 3-
Cys 18 • His 12; P h e
Try! · Tyr 13 Try 4 · Tyr 10 Try 4 · Tyr 10 Try 4 · Tyr 4 r
is
* Tryo * Tyr 24
0.007 0.006 0.007 0.015 0.015
c
Calculated
0
0.01 0.01 d (0.007) 0.014 d (0.0097) 0.01 d (0.006) 0.03 d (0.026) 0.0051 0.011 0.01 0.02 d (0.014) 0.06
°From A. D. McLaren and O. Hidalgo-Salvatierra, Photochem. Photobiol. 3 , 349(1964). b ®ENz c
€
X Vi ¿ &il*ENz (see Table 5-1 and Section 5-4). The quantum yield for carboxypeptidase has been redetermined by R. Piras and B. L. Vallee [Biochemistry 6, 2269 (1967)] to be 0.0049 which now brings the calculated value (0.01) within about a factor of two of the observed value. Newer data (cited by Piras and Vallee) indicate that carboxypeptidase contains no S—S bridges. d Often a much closer agreement is obtained between the observed and calculated quantum yields if only the cystine residues are used in the calculations instead of using all of the amino acids that absorb UV light. This is permissible as a special condition of the formulation but suggests that the statement by McLaren and Hidalgo-Salvatierra (citation in footnote a) that "the site of quantum absorption and the site of photochemical reaction in proteins are one and the same and that it is not necessary to invoke a mechanism of migration of absorbed energy from one kind of chromophore to another within an absorbing molecule" is not valid for all proteins, for all proteins. —
94
5.
PHOTOCHEMISTRY OF AMINO ACIDS AND PROTEINS
O n e may take exception with certain of the a s s u m p t i o n s used by M c L a r e n and c o - w o r k e r s in developing their theory but these p r o b ably would not c a u s e o r d e r of magnitude (ten fold) e r r o r s . T h e calculated q u a n t u m yields for seven e n z y m e s agree with the o b served q u a n t u m yields within a factor of t w o and this m u s t be considered to support their theory. H o w e v e r , the d a n g e r of theories is that they may be a c c e p t e d as universal and therefore stifle future research. P e r h a p s an equally significant contribution that this theory can m a k e to the photochemistry of proteins is the fact that it d o e s have several marked exceptions. A u g e n s t e i n ' s theory deals prin cipally with those proteins which d o not closely fit the theory of M c L a r e n , and the work of Augenstein and c o - w o r k e r s oifers c o n vincing data in support of the importance of energy migration in the photochemical inactivation of certain proteins. W e may conclude that both theories are correct but for different proteins. T h e r e must be unique structural features or unique amino acid s e q u e n c e s in s o m e proteins that allow the a m i n o acids to be independent absorbers and for photochemical inactivation e v e n t s to occur apparently at r a n d o m , while different structural features in other proteins give rise to the opposite results (i.e., n o n r a n d o m photochemical events and the involvement of energy migration in the inactivation of enzymes). Although it has long been k n o w n that U V has a deleterious effect upon proteins, we still have very little understanding of the funda mental chemical mechanism(s) involved. In fact, our knowledge concerning the photochemistry of the individual amino acids is grossly inadequate. A l s o , there is evidence that not all of the mole cules of a particular amino acid within a protein h a v e the s a m e photochemical sensitivity. Until w e h a v e a b e t t e r u n d e r s t a n d i n g of the primary photochemistry of the individual amino acids (singly and in peptides), w e c a n n o t h o p e to u n d e r s t a n d properly the photochemical m e c h a n i s m s involved in the inactivation of proteins. Interest in the photochemistry of the amino acids and proteins has been o v e r s h a d o w e d in recent years by the phenomenal surge in research activity on the photochemistry of the nucleic acids. O u r knowledge of the photochemistry of the nucleic acids now far ex c e e d s our knowledge of the photochemistry of the proteins. A n a d e q u a t e knowledge of both is n e c e s s a r y , h o w e v e r , before w e c a n
GENERAL REFERENCES
95
properly u n d e r s t a n d t h e m e c h a n i s m of a c t i o n of U V o n cells. T h e paucity of k n o w l e d g e of t h e m o l e c u l a r m e c h a n i s m s i n v o l v e d in t h e p h o t o c h e m i s t r y of t h e p r o t e i n s (and a m i n o acids) m a k e s it a very attractive field for future r e s e a r c h .
GENERAL REFERENCES R. B. Setlow, A relation between cystine content and ultraviolet sensitivity of pro teins. Biochim. Biophys.Acta 16, 444 (1955). R. B. Setlow, Radiation studies on proteins and enzymes. Ann. N.Y. Acad. Sei. 59, 469(1955). A. D. McLaren and D. Shugar, "Photochemistry of Proteins and Nucleic Acids." Pergamon Press, Oxford, 1964. A. D. McLaren and O. Hidalgo-Salvatierra, Quantum yields for enzyme inactivation and the amino acid composition of proteins. Photochem. Photobiol. 3 , 349 (1964). Theory: Random destruction of amino acids causes inactivation. Energy transfer considerations are not necessary to explain inactivation. L. Augenstein and P. Riley, The inactivation of enzymes by ultraviolet light. IV. The nature and involvement of cystine disruption. Photochem. Photobiol. 3 , 353 (1964); see also, ibid. 6, 423 (1967). Theory: Inactivation is caused by the disruption of specific cystine residues and of hydrogen bonds responsible for the spacial integrity of the active center of the enzyme. Energy transfer considerations are important.
5-1.
Introduction Relative Photochemical Sensitivity of the Amino Acids General Comments on the Photochemistry of Proteins . Photochemical Inactivation of Enzymes General References
85 86 88 91 95
INTRODUCTION
During the past 10-15 years, general interest in photobiology has strongly emphasized the nucleic acids, although currently there is r e n e w e d interest in p h o t o n effects on proteins. M o s t of the d a t a on the photochemical reactions of the amino acids, h o w e v e r , w e r e gathered s o m e years ago without the benefit of m o n o c h r o m a t i c light sources, the availability of labeled amino acids, or of m o d e r n m e t h o d s of c h r o m a t o g r a p h y and electrophoresis for the separation of the p h o t o p r o d u c t s . T h e reported q u a n t u m yields for proteins are sometimes in error due to inadequate knowledge (at that time) of the molecular weight and amino acid composition of the particular proteins u n d e r investigation. T h e ready availability of synthetic polyamino acids now provides an excellent opportunity for further exciting work on the photochemistry of the amino acids especially as regards possible energy migration and neighbor interactions. O u r understanding of the effects of U V o n cells requires an u n d e r s t a n d ing of the chemical modifications p r o d u c e d in proteins by U V . T h e finding that proteins and D N A interact photochemically within the cell (Section 4-9) further necessitates a renaissance in studies on the photochemistry of amino acids and proteins. 85
5.
86
5-2.
RELATIVE
PHOTOCHEMISTRY OF AMINO ACIDS A N D PROTEINS
PHOTOCHEMICAL SENSITIVITY OF THE
AMINO
ACIDS
Since light must be a b s o r b e d before photochemistry will occur, it is reasonable to a s s u m e that those amino acids that are transparent to the wavelengths normally used in photobiology (wavelengths greater than 2 4 0 0 Ä ) will undergo little or no p h o t o c h e m i s t r y at these wavelengths. T h i s then largely eliminates the aliphatic a m i n o acids from our consideration. T h e strong absorption of wavelengths above 2 4 0 0 A by the aromatic amino acids would certainly suggest their photochemical importance, although it should be recalled that absorbed light need hot necessarily result in p h o t o c h e m i s t r y (Sec tion 1 - 2 ) . Before continuing with the photochemistry of the aromatic amino acids, let us consider two c h r o m o p h o r e s that are not present in monomeric amino acids but are present in proteins: peptide linkages and disulfide bridges. T h e absorption spectrum for peptide b o n d s shows a peak in the region of 1 8 0 0 - 1 9 0 0 A which d e c r e a s e s essen tially to zero by about 2 4 0 0 Ä . Peptide b o n d s are b r o k e n by ab sorption at these wavelengths. Therefore, if peptide bond breakage is to be minimized, wavelengths below 2 4 0 0 Ä must be filtered out of the light source. Even at 2 5 3 7 Ä , however, the q u a n t u m yield for peptide bond breakage is a significant value ( T a b l e 5 - 1 ) . T h e amino acids, cysteine and cystine, were considered at one time to be unimportant photochemically since according to their absorption spectra in acid solution they w e r e essentially optically transparent a b o v e 2 3 0 0 A. A t neutral and alkaline p H , h o w e v e r , the absorption by these c o m p o u n d s b e c o m e s appreciable even at wavelengths above 2 5 0 0 Ä . T h i s fact coupled with the high q u a n t u m efficiency for their photochemical alteration m a k e s cystine the most sensitive target affecting protein function. If the molar absorptivity (e) of a c o m p o u n d is a m e a s u r e of the probability that light of a particular wavelength will be absorbed by that c o m p o u n d , and the q u a n t u m yield ( Φ ) is the probability that the absorbed light will cause a chemical change, then the p r o d u c t of these two values (e Χ Φ ) is a m e a s u r e of the photochemical sensi tivity of that c o m p o u n d . Values for these p a r a m e t e r s at 2 5 3 7 A for the most important c h r o m o p h o r e s in proteins are given in T a b l e 5 - 1 . T h u s , w h e n cystine is present in a protein it is the most labile target. Following in decreasing lability are t r y p t o p h a n , phenylalanine,
5-2.
PHOTOCHEMICAL SENSITIVITY OF AMINO ACIDS
TABLE 5-1.
87
T H E RELATIVE PHOTOCHEMICAL LABILITY OF AMINO ACIDS
AT 2537 A« b
0
Compound
e
φο
(e Χ Φ)
Cystine (—S—S— bond) Tryptophan Phenylalanine Tyrosine Peptide bonds (acetylalanine) Histidine
270 2870 140 320 0.2 0.24
0.13 0.004 0.013 0.002 0.05 <0.03
35.1 11.5 1.8 0.6 0.01 <0.0072
"Data from A. D. McLaren and D. Shugar, "Photochemistry of Proteins and Nucleic Acids," p. 97. Pergamon Press, Oxford, 1964. b e indicates molar absorptivity at 2537 A, or the probability that light at 2537 Λ will be absorbed by the given compound. ""Φ indicates quantum yield (the probability that the absorbed light (2537 Ä) will cause a chemical change). d (e x Φ) indicates a measure of the photochemical sensitivity of a particular amino acid (it is related to the inactivation cross-section, cr, by a constant; Section 6-2).
tyrosine, peptide b o n d s , and histidine. It should be stressed, how ever, that at other wavelengths the U V sensitivity and therefore the relative importance of these targets in the photochemical in activation of a particular protein will change. Most of the early photochemical studies on the amino acids did not use m o n o c h r o m a t i c light and the analysis for photochemical alteration relied upon easily measurable p r o d u c t s such as ammonia, carbon dioxide, acids, b a s e s , sulfur and loss of spectral properties. T h e importance of using m o n o c h r o m a t i c light for photochemical studies has already been stressed (e.g., Section 4-5). Relying only upon end products of destruction such as a m m o n i a , c a r b o n dioxide, etc., very little information can be gained about intermediate reac tions that o c c u r at biologically significant d o s e s of U V . A recent study on the p h o t o c h e m i s t r y of phenylalanine (selec 14 tively labeled at different positions with C ) has s h o w n that w h e n this amino acid is irradiated at the wavelength of m a x i m u m a b s o r p tion of the aromatic residue (2575 Ä) that tyrosine, dihydroxyphenylalanine ( D O P A ) , aspartic acid, benzoic acid, phenyllactic acid, and a high molecular weight melanic polymer are formed. T h e reaction that occurred with the greatest q u a n t u m efficiency w a s the liberation of carbon dioxide by decarboxylation. T h e s e results indicate that there w a s cleavage of side chain c a r b o n b o n d s , and
88
5.
PHOTOCHEMISTRY OF AMINO ACIDS A N D
PROTEINS
since all of the energy was initially absorbed by the b e n z e n e ring, it is therefore reasonable to conclude that energy migration by some mechanism (as yet not understood) must have occurred. T h e direct transmission of energy along — ( C H 2 ) W— would a p p e a r to b e incon sistent with the observation (Section 1-3) that m e t h y l e n e groups affect the absorption spectra of c o m p o u n d s by acting as insulators between aromatic groups. Several other examples of energy migration in peptides and pro teins are known. T h e photochemical decomposition of c a r b o n monoxide-myoglobin o c c u r s with the same q u a n t u m yield regard less of whether the light energy is absorbed by the aromatic amino acids of the protein or the porphyrin ring. W h e n the d y e 1-dimethylaminonaphthalene-5-sulfonyl chloride w a s b o u n d to proteins and the complex irradiated with light that was specifically absorbed by the aromatic residues of the protein it was found that the d y e fluoresced. T h e fluorescence of the d y e could only be explained on the basis of energy transfer from the protein. This type of response has also been shown to result in a d e c r e a s e in the inactivation rate of ir radiated proteins. Energy migration is thought to o c c u r in part through a resonance-transfer mechanism. A fluorescent donor, with a fluorescence spectrum overlapping the absorption spectrum of the energy acceptor, can pass energy to the a c c e p t o r if the dis tance between the two is small (less than 100 Λ) and if the relative orientation b e t w e e n the t w o is suitable (Section 3-7). Since it is possible to distinguish b e t w e e n the spectra of phenylalanine, tyro sine and tryptophan the fluorescence and p h o s p h o r e s c e n c e spectra of proteins can yield information on the direction of transfer of energy b e t w e e n these amino acids. Phenylalanine fluorescence is not observed from most proteins although a tyrosine c o m p o n e n t is always observed. T y r o s i n e fluorescence m a k e s only a small con tribution to the total fluorescence from those proteins containing tryptophan. T h e yields of cystine destruction are m u c h higher in those proteins that contain tryptophan, again emphasizing the im portance of energy transfer in an irradiated protein (Section 5-4). 5-3.
GENERAL
COMMENTS
ON
THE
PHOTOCHEMISTRY
OF
PROTEINS
(a) W h e n proteins are irradiated with U V , both lower and higher molecular weight products are formed. T h e higher molecular weight
5-3.
G E N E R A L C O M M E N T S O N PHOTOCHEMISTRY O F PROTEINS
89
aggregates are m u c h easier to detect, h o w e v e r , and a p p e a r to be produced with the greatest efficiency. While aggregation p r o c e e d s at the same rate u n d e r oxygen or nitrogen at lower d o s e s of U V , at higher d o s e s in the p r e s e n c e of oxygen, the aggregates begin to d e c o m p o s e and the solubility of the irradiated protein therefore begins to increase. (b) Although certain aspects of the p h o t o c h e m i s t r y of proteins are dependent upon w h e t h e r they are irradiated in the p r e s e n c e of oxygen (see above), the q u a n t u m yield for inactivation of an e n z y m e does not d e p e n d upon the a t m o s p h e r e during irradiation. (c) Irradiated proteins are much more susceptible to enzymatic digestion. T h i s , coupled with the fact that heat d e n a t u r e d proteins are in general more susceptible to e n z y m a t i c digestion, indicates that U V c a u s e s the denaturation of proteins. It has also b e e n o b served that following d o s e s of U V (which c a u s e no serious loss in enzymatic activity) the e n z y m e s are m u c h more sensitive to subse quent heat inactivation. At least t w o m e c h a n i s m s could explain this observation. If a peptide bond were b r o k e n but the b r o k e n ends were held in place by the hydrogen b o n d s involved in the sec ondary and tertiary structures of the protein, then the molecule would be enzymatically active until gently heat treated. If a disulfide bridge were broken by U V , then it might require less heat to dis organize the hydrogen bonds of the molecule and in the a b s e n c e of the directional influence of the disulfide bridge, the original hydrogen b o n d s would be less likely to reform on cooling than they would have prior to irradiation. (d) N e a r room t e m p e r a t u r e , the q u a n t u m yield for the inactiva tion of an e n z y m e does not show any m a r k e d d e p e n d e n c y upon the temperature during irradiation. H o w e v e r , at t e m p e r a t u r e s below room t e m p e r a t u r e there is a continuous d e c r e a s e (as the tempera ture decreases) in the q u a n t u m yield; while as the t e m p e r a t u r e in creases a b o v e r o o m t e m p e r a t u r e , there is a c o n t i n u o u s increase in q u a n t u m yield. W e have c o m m e n t e d previously on the effect of temperature and environment upon the a b s o r b a n c e of various com pounds ( C h a p t e r 1) and on the photochemical reactivity of D N A (Chapter 4). Such considerations may possibly be invoked to explain the effect of t e m p e r a t u r e on the photochemical reactivity of proteins. (e) T h e q u a n t u m yields for the U V inactivation of e n z y m e s irradiated in the dry state and in a q u e o u s m e d i u m d o not differ significantly. This suggests that the m e c h a n i s m s involved in the
90
5.
PHOTOCHEMISTRY OF AMINO ACIDS A N D
PROTEINS
two cases are not radically different, but this may well be an over simplification. (f) T h e q u a n t u m yield for proteins varies with the wavelength of irradiation. T h e explanation for this is that the action s p e c t r u m for the inactivation of an e n z y m e can be resolved into the contributions by the various c h r o m o p h o r e s . At 2537 Ä , the q u a n t u m yield for the inactivation of trypsin can be adequately accounted for by assigning the major role to cystine, but at wavelengths a b o v e this value the aromatic amino acids play a more important role and at wavelengths below 2537 A peptide b o n d s play an important role. (g) T h e q u a n t u m yield (at 2537 Ä) for the inactivation of an e n z y m e is roughly proportional to its cystine content. T h i s is con sistent with the data cited in T a b l e 5-1 and will be more fully dis cussed in Section 5-4. (h) T h e q u a n t u m yield for the inactivation of an e n z y m e is roughly T A B L E 5-2.
SOME CHANGES PRODUCED IN PROTEINS FOLLOWING EXPOSURE TO
Property
U V
Change
Color Solubility in water
Darkens Decreases; then increases
Sensitivity to heat
Usually increases
Optical rotation Change in absorption
Increase in levorotation Increases
Digestibility by trypsin Exposure of SH groups Exposure of acid groups
Increases Increases Increase; pH change
Electrophoretic pattern Ultracentrifugation Immunological effects
Heterogenization Increased broadening of sedimentation bands Destruction of antigenic properties; formation of new ones
Possible meaning Photooxidation Aggregation, then degradation Hydrophobic groups exposed; chain breaks Structural changes Depolymerization and photooxidation; formation of new chromophores Opening up of molecule Opening up of molecule Photooxidation to form more phenols; breakage of peptide bonds Degradation and reaggregation Molecular weight hetero geneity Altered amino acid residues
"Modified from A. C. Giese, Physiol. Rev. 30, 431 (1950).
5-4.
PHOTOCHEMICAL I N A C T I V A T I O N O F E N Z Y M E S
91
proportional to the reciprocal of its molecular weight. A precise explanation for this observation is not available, h o w e v e r , it has been pointed out that the cystine content of proteins is roughly proportional to the reciprocal of their molecular weights. (i) In most cases the q u a n t u m yields for the inactivation of the nucleic acids are less than those for proteins, yet the molar a b s o r b tivity for the proteins are m u c h lower t h a n for t h e nucleic acids. M c L a r e n has o b s e r v e d that the p r o d u c t of the molar absorptivity (e) and q u a n t u m yield (Φ) for proteins at 2537 A is approximately equal to that for the nucleic acids. T h e significance of this o b s e r v a tion remains to be evaluated but at face value it would imply that proteins may play a larger role in biological inactivation than most of the nucleic acid chemists would c a r e to admit. (j) T h e q u a n t u m yields for the inactivation of e n z y m e s usually vary with p H but there a p p e a r s to be no correlation with the iso electric point of the e n z y m e . T h e overall charge on the molecule would therefore appear to be of less importance than the charge on some specific amino acid residue(s). (k) O t h e r changes noted in proteins following e x p o s u r e to U V are listed in Table 5-2. 5-4.
PHOTOCHEMICAL
INACTIVATION OF
ENZYMES
T w o different theories of the m e c h a n i s m s involved in the U V inactivation of e n z y m e s h a v e d e v e l o p e d o v e r a period of y e a r s . M c L a r e n and c o - w o r k e r s h a v e a s s u m e d , as a first approximation, that the alteration of any amino acid residue c a u s e s inactivation of an e n z y m e . If the quantity (e x Φ) for each amino acid (which is a measure of the photochemical sensitivity of each amino acid; see T a b l e 5-1) is multiplied by the n u m b e r of such amino acids (n) in the protein and the sum of these is divided by the molar a b sorptivity of the e n z y m e , one arrives at a calculated q u a n t u m yield for the inactivation of the e n z y m e which in s o m e c a s e s is very close to the observed q u a n t u m yield. Of the ten proteins listed in T a b l e 5-3, the calculated q u a n t u m yields for seven of these are within a factor of 2 of the experimentally determined values. T h e values for c a r b o x y p e p t i d a s e , pepsin, and insulin do not a d e q u a t e l y fit the hypothesis. T h e authors themselves h a v e pointed u p certain criticisms of
92
5.
PHOTOCHEMISTRY OF AMINO ACIDS AND PROTEINS
these calculations. T h e r e is no a s s u r a n c e that either the molar absorptivity or the q u a n t u m yield for a particular amino acid (at a given p H and wavelength) is the s a m e in a protein as it is in free solution; in fact, there is s o m e evidence that these values change w h e n the amino acid is incorporated into a protein. Also it is k n o w n that all residues of a particular amino acid in a given protein are not of equal importance to enzymic function. F o r those few proteins studied, their a b s o r b a n c e at 2537 Á d o e s not differ markedly from that for an equivalent mixture of the free amino acids although greater differences exist at other wavelengths. This fact, plus the rather close agreement b e t w e e n the calculated and determined q u a n t u m yields cited in T a b l e 5-3 would suggest that u n d e r certain conditions the amino acid residues are, in fact, independent absorbers unaffected by their neighbors. M c L a r e n and co-workers therefore state that energy transfer a m o n g c h r o m o p h o r e s within molecules of e n z y m e s need not be invoked in o r d e r to account for photochemical inactivation. Augenstein and associates, h o w e v e r , take particular exception to this conclusion. T h e y have developed a theory that e n z y m e s are inactivated by U V as a c o n s e q u e n c e of the disruption of specific cystine residues and of hydrogen b o n d s responsible for the spatial integrity of the active center of the e n z y m e . T h e i r theory e m p h a s i z e s the importance of energy migration in this p r o c e s s . C o n s i s t e n t with these postulates are the observations that the photochemical d a m a g e in the e n z y m e ribonuclease is n o n r a n d o m ; at least t w o and p e r h a p s three of the four constituent cystines must b e disrupted before activity is lost, i.e., the most photosensitive cystines are not critical for enzymic activity. Similarly, in both trypsin and l y s o z y m e the integrity of the m o s t photosensitive cystines d o e s not a p p e a r to b e critical for the retention of enzymic potential. In insulin, h o w e v e r , all three cystines a p p e a r to be crucial for activity and h a v e approxi mately equal photosensitivities. T h e s e differences in sensitivity of cystines in different proteins must d e p e n d specifically u p o n energy transfer a n d / o r chemical interactions b e t w e e n the c h r o m o p h o r i c groups. If q u a n t u m yields are calculated on the basis of those q u a n t a absorbed only in the cystines, values for cystine destruction in proteins are obtained which are a b o u t 5 to 8 times greater than those observed in the model c o m p o u n d s , cystine and oxidized glutathione. Cystine destruction is m u c h greater in those proteins which contain
5-4.
93
PHOTOCHEMICAL INACTIVATION OF ENZYMES
tryptophan. T h e r e is s o m e evidence that the most photosensitive cystines lie close to t r y p t o p h a n residues as a c o n s e q u e n c e of the tertiary structure of the e n z y m e rather than of its primary structure. W e seem to be faced with t w o opposing theories c o n c e r n i n g the involvement of energy transfer in the U V inactivation of e n z y m e s .
T A B L E 5-3.
CALCULATED AND K N O W N QUANTUM YIELDS FOR ENZYME
INACTIVATION BY UV (2537 A)°
Quantum yield Protein
Chromophores
Found
1. Carboxypeptidase 2. Chymotrypsin
Cys 2- His 8- Phe 15 •Try 6 • Tyr 20 Cys 5- His 2 · Phe 6- Try 7 · Tyr 4
0.00 l 0.005
3. Lysozyme
Cys 5- His!- Phe 3- Try 8 · Tyr 2
0.024
4. Pepsin
Cys 3- His 2 · Phe 9- Try 4 · Tyr 16
0.002
5. Ribonuclease
Cys 4- His 4 · Phe 3- Try 0 · Tyr 16
0.027
6. 7. 8. 9.
Cys 0CysoCysoCys 6-
Subtilisin A Subtilisin Β Japanese Nagarse Trypsin
10. Insulin
His 5His 6His 6His x-
Phe 4Phe 3Phe 3Phe 3-
Cys 18 • His 12; P h e
Try! · Tyr 13 Try 4 · Tyr 10 Try 4 · Tyr 10 Try 4 · Tyr 4 r
is
* Tryo * Tyr 24
0.007 0.006 0.007 0.015 0.015
c
Calculated
0
0.01 0.01 d (0.007) 0.014 d (0.0097) 0.01 d (0.006) 0.03 d (0.026) 0.0051 0.011 0.01 0.02 d (0.014) 0.06
°From A. D. McLaren and O. Hidalgo-Salvatierra, Photochem. Photobiol. 3 , 349(1964). b ®ENz c
€
X Vi ¿ &il*ENz (see Table 5-1 and Section 5-4). The quantum yield for carboxypeptidase has been redetermined by R. Piras and B. L. Vallee [Biochemistry 6, 2269 (1967)] to be 0.0049 which now brings the calculated value (0.01) within about a factor of two of the observed value. Newer data (cited by Piras and Vallee) indicate that carboxypeptidase contains no S—S bridges. d Often a much closer agreement is obtained between the observed and calculated quantum yields if only the cystine residues are used in the calculations instead of using all of the amino acids that absorb UV light. This is permissible as a special condition of the formulation but suggests that the statement by McLaren and Hidalgo-Salvatierra (citation in footnote a) that "the site of quantum absorption and the site of photochemical reaction in proteins are one and the same and that it is not necessary to invoke a mechanism of migration of absorbed energy from one kind of chromophore to another within an absorbing molecule" is not valid for all proteins, for all proteins. —
94
5.
PHOTOCHEMISTRY OF AMINO ACIDS AND PROTEINS
O n e may take exception with certain of the a s s u m p t i o n s used by M c L a r e n and c o - w o r k e r s in developing their theory but these p r o b ably would not c a u s e o r d e r of magnitude (ten fold) e r r o r s . T h e calculated q u a n t u m yields for seven e n z y m e s agree with the o b served q u a n t u m yields within a factor of t w o and this m u s t be considered to support their theory. H o w e v e r , the d a n g e r of theories is that they may be a c c e p t e d as universal and therefore stifle future research. P e r h a p s an equally significant contribution that this theory can m a k e to the photochemistry of proteins is the fact that it d o e s have several marked exceptions. A u g e n s t e i n ' s theory deals prin cipally with those proteins which d o not closely fit the theory of M c L a r e n , and the work of Augenstein and c o - w o r k e r s oifers c o n vincing data in support of the importance of energy migration in the photochemical inactivation of certain proteins. W e may conclude that both theories are correct but for different proteins. T h e r e must be unique structural features or unique amino acid s e q u e n c e s in s o m e proteins that allow the a m i n o acids to be independent absorbers and for photochemical inactivation e v e n t s to occur apparently at r a n d o m , while different structural features in other proteins give rise to the opposite results (i.e., n o n r a n d o m photochemical events and the involvement of energy migration in the inactivation of enzymes). Although it has long been k n o w n that U V has a deleterious effect upon proteins, we still have very little understanding of the funda mental chemical mechanism(s) involved. In fact, our knowledge concerning the photochemistry of the individual amino acids is grossly inadequate. A l s o , there is evidence that not all of the mole cules of a particular amino acid within a protein h a v e the s a m e photochemical sensitivity. Until w e h a v e a b e t t e r u n d e r s t a n d i n g of the primary photochemistry of the individual amino acids (singly and in peptides), w e c a n n o t h o p e to u n d e r s t a n d properly the photochemical m e c h a n i s m s involved in the inactivation of proteins. Interest in the photochemistry of the amino acids and proteins has been o v e r s h a d o w e d in recent years by the phenomenal surge in research activity on the photochemistry of the nucleic acids. O u r knowledge of the photochemistry of the nucleic acids now far ex c e e d s our knowledge of the photochemistry of the proteins. A n a d e q u a t e knowledge of both is n e c e s s a r y , h o w e v e r , before w e c a n
GENERAL REFERENCES
95
properly u n d e r s t a n d t h e m e c h a n i s m of a c t i o n of U V o n cells. T h e paucity of k n o w l e d g e of t h e m o l e c u l a r m e c h a n i s m s i n v o l v e d in t h e p h o t o c h e m i s t r y of t h e p r o t e i n s (and a m i n o acids) m a k e s it a very attractive field for future r e s e a r c h .
GENERAL REFERENCES R. B. Setlow, A relation between cystine content and ultraviolet sensitivity of pro teins. Biochim. Biophys.Acta 16, 444 (1955). R. B. Setlow, Radiation studies on proteins and enzymes. Ann. N.Y. Acad. Sei. 59, 469(1955). A. D. McLaren and D. Shugar, "Photochemistry of Proteins and Nucleic Acids." Pergamon Press, Oxford, 1964. A. D. McLaren and O. Hidalgo-Salvatierra, Quantum yields for enzyme inactivation and the amino acid composition of proteins. Photochem. Photobiol. 3 , 349 (1964). Theory: Random destruction of amino acids causes inactivation. Energy transfer considerations are not necessary to explain inactivation. L. Augenstein and P. Riley, The inactivation of enzymes by ultraviolet light. IV. The nature and involvement of cystine disruption. Photochem. Photobiol. 3 , 353 (1964); see also, ibid. 6, 423 (1967). Theory: Inactivation is caused by the disruption of specific cystine residues and of hydrogen bonds responsible for the spacial integrity of the active center of the enzyme. Energy transfer considerations are important.