The labelling of proteins by irradiation in tritiated water

432 BBA

BIOCI-IIMICA :ET BIOPHYSICA ACTA

26825

T H E LABELLING OF PROTEINS BY IRRADIATION IN T R I T I A T E D WATER L E O A. H O L T AND B R I A N M I L L I G A N

Division of Protein Chemistry, CSIRO, Parkville (Melbourne), Victoria 3052 (Australia) (Received N o v e m b e r i 6 t h , i971)

SUMMARY

I. Tritium is irreversibly bound by myoglobin, S-carboxymethyllysozyme, lysozyme and wool during ultraviolet irradiation in the presence of tritiated water. In each case the main site of labelling, determined by radioassay of the amino acids liberated by enzyme hydrolysis, is the tryptophan residue. Tyrosine, phenylalanine, histidine and S-carboxymethylcysteine residues are labelled to a lesser extent. 2. Labelling probably occurs as the result of various transient species, produced by photolysis, abstracting tritium from the solvent. The extent of labelling at different sites does not necessarily reflect the relative photolytic susceptibilities of these sites because, in the case of tryptophan residues at least, the rate of photodecomposition exceeds the rate of labelling.

INTRODUCTION

Electron spin resonance spectroscopy has frequently been used to determine the primary sites of photolysis of proteins. Generally, ultraviolet irradiation of proteins gives two types of signal, ascribed to free radical species derived from cystine residues and from one or more of the aromatic amino acid residues. The subject has been covered in a recent review by Vladimirov et al. 1. The technique of flash photolysis has also been used to study the transient species produced during irradiation of proteins (see e.g. ref. 2). Holt and Leach 3 have used an alternative approach, viz. the identification of those amino acid residues which become iadioaetively labelled during irradiation of the protein in tritiated water. These labelled amino acids were believed to arise when free radical species, produced by photolytic dissociation of hydrogen atoms, abstracted tritium from the solvent. In their experiments, Holt and Leach exposed wool, immersed in tritiated water, to sunlight and then hydrolysed the protein to its constituent amino acids with boiling HC1. This procedure was unsatisfactory because it destroyed tryptophan, one 6f the most likely sites of photolysis. Also, acid hydrolysis of proteins in the presence of tritium ious leads to the incorporation of radioactivity by many of the resulting amino acids4; this probably explains why a number of labelled amino acids were isolated in a control experiment in which the irradiation step was omitted. In the present work the approach of Holt and Leach 3 has been used to investigate the occurrence of events leading to exchange of hydrogen atoms at C-sites Biochim. Biophys. Acta, 264 (I972) 432-439

LABELLING OF PROTEINS BY IRRADIATION IN

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433

with the solvent during irradiation of proteins with low-energy ultraviolet light. Four proteins, viz. myoglobin, lysozyme, S-carboxymethyllysozyme and wool, were irradiated in the presence of tritiated water and then hydrolysed with a mixture of the three proteolytic enzymes, pronase, prolidase and leucine aminopeptidase. This method of hydrolysis is preferable to acid hydrolysis in the present application because tryptophan, in addition to most other amino acids, is obtained in good yield. A further advantage is that enzyme hydrolysis of proteins that have been treated with tritiated water, but not irradiated, yields no irreversibly-labelled amino acids; therefore correction factors are not required. The sites of labelling of the irradiated proteins were determined by radioassay of the amino acids separated from enzyme hydrolysates by ion-exchange chromatography. The extent of photodecomposiLion of tryptophan residues, the most photosensitive residues present, was also determined in order to establish the relative rates of labelling and photodecomposition. EXPERIMENTAL

Materials Sperm whale myoglobin (Koch-Light) was freed from haem by the method of Teale 5. Egg white lysozyme (Sigma) was reduced and carboxymethylated according to the method of O'Donnell and Thompson ~. The plain-weave, light-weight wool fabric was woven from merino wool (64's quality) ; it was extracted successively with light petroleum, ethanol and water before use. Pronase AF (Kaken Chem. Co., Tokyo) was pulified by fractional precipitation from aqueous acetone followed by chromatography on DEAE-cellulose. The leucine aminopeptidase and prolidase were commercial samples from P - L Biochemicals (Milwaukee, Wisc.) and Miles Labs (Kankankee, Ill.), respectively. Tritiated water (5 Ci/ml) from the Radiochemical Centre (Amersham, England) was diluted to IOO mCi/ml with deionized water before use. Irradiations These were performed in a 2o-ml cell surrotmding a jacketted Ioo-W Hanovia medium-pressure mercury lamp. Water at 20 °C was circulated through the Pyrex jacket surrounding the lamp; only wavelengths > 295 nm were transmitted. Either nitrogen or air was passed through the cell during irradiation. Solutions of myoglobin or lysozyme (ioo rag) were prepared for irradiation by dissolving the protein in 0.o2 M ammonium acetate buffer (pH 4.2, IO ml) and then adding an equal volume of tritiated water (IOOmCi/ml). Solutions of S-earboxymethyllysozyme were prepared indirectly by dissolving the protein in 8 M urea, adjusting to pH 4.2 and dialysing against o.02 M ammonium acetate (pH 4.2); tritiated water was then added as before. These protein solutions were irradiated for times up to 2 h. Before irradiation the wool fabric (approx. I.O g) was wet with a warm aqueous solution of non-ionic detergent (o.oi %), rinsed thoroughly in deionized water and then blotted to remove excess moisture. The weight of water still present (approx. 0.5 g) was determined by weighing. An equal weight of tritiated water (IOO mCi/ml) was then added and the wool squeezed between glass plates to facilitate thorough mixing. The impregnated fabric was wrapped around the water jacket of the lamp and (in some experiments) nitrogen was passed through the cell for 2 h before irradiaBiochim. Biophys. Acta, 264 (i972) 432-439

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L, A. HOLT, B. MILLIGAN

tion was begun. After irradiation for 2 h the fabric was turned over and its other face exposed similarly. The irradiated soluble proteins were dialysed against water at 4 °C until negligible radioactivity was found in the dialysate (24-48 h); the irradiated wool fabric was washed with water for 24 h.

Enzyme hydrolysis Myoglobin and S-carboxymethyllysozyme samples were hydrolysed directly but lysozyme and wool were first reduced and carboxymethylated by the method of O'Donnell and Thompson 6. The proteins were incubated with pronase (2 % on the weight of protein) at pH 8.0 for 24-48 h at 4 ° °C. Prolidase (2 %) and leucine aminopeptidase (2 %) were then added and the mixtures were incubated for a further 24 h, as described previously 7.

Analysis of enzyme hydrolysates Hydrolysates were subjected to ion-exchange chromatography on a column (45 cm × 2 cm diameter) containing Technicon chromobeads (Type C), using pyridineacetic acid buffers (pH 3.26, 4.26 and 5.28, successively) at 50 °C. An aliquot (I.O ml) from each alternate fraction (5 ml) was evaporated and the residue dissolved in 0.2 M ammonia solution for radioassay. After addition of dioxan-based scintillation mixture, the radioactivity was measured using a Packard Tri-Carb liquid scintillation spectrometer (Series 314 EX). Enzyme hydrolysates were also analysed using a Beckman-Spinco amino acid analyzer (Model 12o B). The basic amino acids were separated on a Io-cm column instead of the customary 5-cm column to improve the resolution of tryptophan from lysine. In one experiment the tryptophart contents of a series of irradiated myoglobin samples were determined directly (without prior hydrolysis) using the colorimetric procedure described by Opienska-Blauth et al. 8.

Distribution of labels in tritiated tryptophan Non-labelled tryptophan (20.0 rag) was dissolved by warming in a solution of labelled tryptophan (approx. o.I rag) in 0. 5 M acetic acid (1.5o rrd). Formaldehyde (3.3 %, 0.2 rrd) was added and the mixture was kept at 40 °C for 1. 5 h and then at 5 °C for a further 3 h. The resulting precipitate of 1,2,3,4-tetrahydro-/~-carboline-3carboxylic acid 9 was collected by centrifugation, the supernatant being retained for separate radio-assay. The precipitate was washed twice with 0. 5 M acetic acid and then twice with acetone and dried in vacuo. Yields of the carboline in a total of six experiments ranged from 91% to 97 %. A portion (approx. 4 rag) was dissolved in o.I M NaOH (0.2 ml) for radioassay. The original supernatant was distilled at approx. 50 °C using a cold-finger distillation apparatus and an aliquot (0.2 ml) of the distillate radioassayed. Internal standardization was used to correct for quenching in both samples. The proportion of radioactivity in the distillate, relative to the total activity, provides a measure of the proportion of labels originally present in the 2-position ot the labelled tryptophan. RESULTS AND DISCUSSION Photolysis of a protein by low-energy ultraviolet irradiation (,~ > 295 nm) is most likely to produce a free radical species from an amino acid residue by homolytic Biochim. Biophys. Acta, 264 (1972) 432-439

L A B E L L I N G OF P R O T E I N S BY IRRADIATION IN

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435

fission of a C-H, N-H, 0 - H or S-H bond, since these are the weakest bonds present. This radical may then undergo further reactions leading to photodecomposition products. Alternatively, it may abstract a hydrogen atom from another species, usually the solvent, and thereby revert to the original amino acid residue. If tritiated water is used as solvent, then this residue will become radioactively labelled. Tritium incorporation may also occur via excited states or ionic species. Photoexcitation oi indoles, for example, greatly increases their ease of protonation 1° and it is conceivable that tritium incorporation could occur as the result of tritium/hydrogen exchange at sites rendered more easily ionizable by photoexcitation. Grossweiner and Usui ~ have shown that photoionization of tryptophan residues occurs during irradiation of aquous solutions of lysozyme. They postulated that some of the tryptophan ions revert to tryptophan residues by reaction with water followed by electron capture. Thus labelling of proteins in tritiated water could involve any one of several types of transient species produced by irradiation. Only those labels introduced at C-sites will be stable to back-exchange, because those introduced at accessible N-, O- or S-sites freely exchange with water. Initial experiments showed that irradiation of myoglobin in tritiated water, in an atmosphere of nitrogen, led to the incorporation of conveniently measurable amounts of activity in 30 rain. Samples of the protein, both before and after irradiation, were subjected to complete enzyme hydrolysis and then analysed. Comparison of the analyses (see Table I) suggests that some photodecomposition of histidine, tryptophan and methionine residues occurred. The small increase in aspartic and glutamic acid contents caused by irradiation may be due to deamination of asparagine TABLE

I

AMINO ACID ANALYSES OF ENZYME HYDROLYSATES OF MYOGLOBIN B~FORE AND AFTER IRRADIATION A m i n o acid a n a l y s e s are g i v e n as residues per m o l e c u l e ; residues are n o r m a l i z e d t o Leu = 18 residues per m o l e c u l e . I r r a d i a t i o n w a s carried o u t in t r i t i a t e d w a t e r for 3o rain in a n a t m o s p h e r e of nitrogen.

A mino acid

Theory *

Before irradiation

After irradiation

Lys His Arg Trp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe

I9 12 4 2 6 5 6 14 4 II 17 8 2 9 18 3 6

19.2 12.o 4 .1 1.85 4.9 . . ** 12.8 4.1 lO. 7 16.1 7.6 2.1 8.9 18.o 3.2 6.o

t9.2 11. 3 4 .0 1.5o 6.6 .

. ** 14. 5 4.2 Io.6 16. 7 8.2 1.6 9 .0 18.o 3.o 5.8

"Given by Edmundson n. * * T h r e o n i n e a n d serine w e r e n o t r e s o l v e d f r o m a s p a r a g i n e a n d g l u t a m i n e in t h e s e a n a l y s e s .

Biochim. Biophys. Acta, 264 ( i 9 7 2 ) 4 3 2 - 4 3 9

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L. A, HOLT, B. MILLIGAN

and glutamine. Fig. I shows an ion-exchange chromatogram of the labelled amino acids in the enzyme hydrolysate. Tryptophan was the most highly radioactive component; phenylalanine, tyrosine and histidine were also labelled, but to a lesser extent. Other small radioactive peaks present were probably photodecomposition products of these labelled amino acids or their peptides. Analysis of a sample ot myoglobin that had been irradiated for only 12 min gave essentially the same distribution of radioactivity, although at a lower level.

Trp

pH 3 26.

0

~

--

pH 4 . 2 6 - -

pH 5 . 2 8 ~

Tyr

His

r 5O

f00 FRACTION NUMBER

150

200

Fig. i. I o n - e x c h a n g e c h r o m a t o g r a m of r a d i o a c t i v e c o m p o n e n t s in an e n z y m e h y d r o l y s a t e of irr a d i a t e d myoglobin. I r r a d i a t i o n c o n d i t i o n s : t r i t i a t e d w a t e r (5o mCi/ml), p H 4.2, 3 ° min.

Examination of the labelled tryptophan isolated from myoglobin after irradiation for 3o min showed that 27% of the total labels present were located in the 2position. This was done by conversion of the tryptophan to 1,2,3,4-tetrahydro-flcarboline-3-carboxylic acid by reaction with formaldehydeg; any tritium label originally present in the 2-position is converted into tritiated water. The distribution of the remaining labels was not determined.

~ ,

N 2j~H

COOH NHz

HCHO

I

~

~

COOH

H

The ease of labelling of tryptophan residues, relative to that of other susceptible residues in myoglobin, is seen very clearly if the activity is expressed in relation to the number of residues present initially (see Table II). Of the two tryptophan residues originally present, o.5 were lost by photodecomposition whereas a total of only o.186 tritium labels were introduced into the residual tryptophan. Thus it appears that the transient species produced by photolysis of tryptophan residues react with water to regenerate the original tryptophan residue at a lower rate than they react in other ways to give decomposition products. This was confirmed by a kinetic study in which myoglobin was irradiated in tritiated water both in the absence and presence of Biochim. Biophys. Acta, 264 (1972) 432-439

437

LABELLING OF PROTEINS BY IRRADIATION IN SHl-[O TABLE II THE

DISTRIBUTION

OF

TRITIUM

LABELS

IN

IRRADIATED

PROTEINS

Irradiation was carried o u t in tritiated w a t e r (5 ° mCi/ml) at p H 4.2 in an a t m o s p h e r e of nitrogen for 3° min.

Protein

A m i n o acicl

Yield (°/o)

Radioactivity incorporated * *

T r i t i u m labels per residue

Myoglobin

Phe Tyr Trp His

97 ioo 75 94

2 7 53 8

o.ool 0.008 0.093 o,oo2

S-Carboxymethyllysozyme

S-CM-Cys Phe Tyr Trp His

78 92 87 76 99

4 2 6 48 I

o,oo2 0.002 0.007 0.o2 9 0.004

Wool

S-CM-Cys Phe Tyr Trp His

60 98 lO 7 ioo 82

4 4 14 9 I

o.oooi 0.0004 0.0009 0.0052 0.0003

*

A/: **

* Of amino acid in an enzyme hydrolysate. As a percentage of the total non-volatile radioactivity in the hydrolysate.

1.0

z

~E

0.5

o. m

•

-n v

1.0

0

30

60

90

120

TIME OF IRRADIATION(MIN)

Fig. 2. (A) The n u m b e r of t r i t i u m labels incorporated per myoglobin molecule during irradiation in tritiated w a t e r in the absence ( O - - O ) and presence ( O - - 0 ) of oxygen. Exchangeable t r i t i u m was first r e m o v e d b y repeatedly adding and evaporating water, t h e n digesting tile protein with pronase and finally e v a p o r a t i n g the digest. (B) The e x t e n t of destruction of t r y p t o p h a n residues in myoglobin during irradiation in the absence ( A - - A ) and presence ( ~ k - - A ) of oxygen.

Biochim. Biophys. Acta, 264 (1972) 432-439

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L. A. HOLT, B. MILLIGAN

oxygen. Fig. 2 shows that appreciable photodecomposition of tryptophan residues occurs during irradiation, especially in the presence of oxygen. Fewer labels were introduced in the presence than in the absence of oxygen, a result to be expected if tryptophan residues are the main sites of labelling. Obviously the transient species arising by photolysis of tryptophan residues decompose faster than they incorporate tritium, especially in the presence of oxygen. The relative rates of decomposition and tritium incorporation by transient species produced from other photosensitive residues in myoglobin may well be different. Thus the relative extents of labelling of the various residues in myoglobin do not necessarily provide a nleasure of the relative photosensitivities of those residues and the present method is unsuitable for this purpose. Chromatographic examination of an enzyme hydrolysate of S-carboxymethyllysozyme that had been irradiated in tritiated water under nitrogen showed that the tryptophan residues were again the main sites of labelling (see Fig. 3). Phenylalanine, tyrosine and S-carboxymethylcysteine were labelled to a lesser extent; no labelling of histidine, which is present in only small amount in this protein, was detected. The extents of labelling and the recoveries of these amino acids from the enzyme hydrolysate are shown in Table II. In another experiment, lysozyme was irradiated as above and then reduced and carboxymethylated prior to enzyme hydrolysis. Fewer radioactive labels were introduced but the distribution was essentially the same as in the case of S-carboxymethyllysozyme. Thus the presence of intact disulphide bonds in the protein affects the extent but not the sites or distribution of labelling. Radioactive labels were also irreversibly incorporated into wool by irradiation in tritiated water in an atmosphere of nitrogen; the level of activity incorporated was only about a tenth of that incorporated into the soluble proteins. After reduction, carboxymethylation and enzyme hydrolysis, ion exchange chromatography revealed the presence of radioactively labelled S-carboxymethylcysteine, phenylalanine,

Trp

•~

pH 326

~--pH

426--

pH 5.28 - ~

Tyr , s-cM cys

^/~

50

I00

I

150

200

FRACTION NUMBER

Fig. 3. Ion-exchange c h r o m a t o g r a m of radioactive components in an enzyme hydrolysate of irradiated S-carboxymethyllysozyme. Irradiation conditions: tritiated water (50 mCi/ml), p H 4.2, 3 ° min.

Biochim. Biophys. Acta, 264 (I972) 432-439

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tyrosine and tryptophan ia addition to a number of unidentified radioactive compounds, presumably photodegradation products. Tryptophan was labelled less heavily than was the case with the other proteins. However, if the radioactivity incorporated is calculated in relation to the number of residues present initially, then it may be seen (see Table II) that tryptophan residues are labelled to a much greater extent than the other susceptible residues; tyrosine residues were the most extensively labelled of the others. A larger percentage of the labels present in the tryptophan were located at the 2 position (45 %) than was the case with the tryptophan from myoglobin irradiated under nitrogen (27 % in the 2 position). As mentioned previously, much less activity was incorporated during irradiation of myoglobin in tile presence ot oxygen than in its absence. Only 13% of the labels present in the tryptophan derived from an enzyme hydrolysate were present in the 2-position, which indicates that this position is more prone to photooxidation than other positions in the molecule. Irradiation of wool in the presence of oxygen led to appreciable destruction of tryptophan and little incorporation of radioactivity in cystine, phenylalanine, tyrosine, tryptophan or histidine residues. Ion-exchange chromatography of an enzyme hydrolysate showed that about half of the total radioactivity was present as a yellow component which was not retarded by the column. This yellow component is of considerable interest in relation to the undesirable yellowing of wool in sunlight and its nature is currently being investigated. ACKNOWLEDGEMENT

We thank Miss V. M. van Leeuwen for competent technical assistance. REFERENCES Y. A. Vladimirov, D. I. R o s h c h u p k i n a n d E. E. Fesenko, Photochem. Photobiol., i i (197 o) 227. L. I. Grossweiner a n d Y. Usui, Photochem. Photobiol., 13 (1971) 195. L. A. H o l t a n d S. J. Leach, Textile Res. J., 35 (1965) 38o. J. Hill a n d S. J. Leach, Biochemistry, 3 (1964) 1814. F. W. J. Teale, Biochim. Biophys. Acta, 35 (1959) 543. I. J. O ' D o n n e l l a n d E. O. P. T h o m p s o n , .dust. J. Biol. Sci., 17 (1964) 963. L. A. Holt, ]3. Milligan a n d C. M. R o x b u r g h , Aust. J. Biol. Sci., 24 (1971) 5o9. J. O p i e n s k a - B l a u t h , M. Charezinski a n d H. Berbec, ,dnal. Biochem., 6 (1963) 69. J. C. Perrone, A. I a c h a n a n d L. A. M. Carneiro, ,dnais..dcad. Brasil. Cienc., 25 (1953) lO7. E. V a n der D o n c k t , in G. Porter, Progress in Reaction Kinetics, Vol. 5, P e r g a m o n Press, Oxford, 197 ° , p. 294. t i A. B. E d m u n d s o n , Nature, 205 (1965) 883. I 2 3 4 5 6 7 8 9 io

Biochim. Biophys. Acta, 264 (1972) 432-439