Fluorescent substances in protein hydrolyzates I. Acid “Hydrolyzates” of individual amino acids

Fluorescent 1. Acid

Substances

“Hydrolyzates”

in Protein of Individ,ual

Hydrolyzates Amino

Acids1

34. LEDV’INA2 AND F. S, LABELLA:

Received

November

19, 1969

Several structural proteins which we have been investigating contain certain fluorescent substances in addition to the normally occurring amino acids. Some of these fluorescent substances are derived from constituent amino acids in the proteins, whereas others are as yet unidentified. In attempting to isolate and characterize the various fluerescent materials, some of which appear to be involved in protein cross-linking, it is necessary tlo distinguish between those occurring naturally and others producecl as artifact’s during protein purificat,ion and acid hydrolysis. In t’his and t.he accompanying paper, we have examined the fluorescence of purified amino acids, before and after treatment with acid, and in several highly purified proteins and peptidca. By examining both the fluoresccncc spectra and t’he fluorescence intensity of various subst’ancesproductbd by the conditions of acid hydrolysis, as well as during separaGon and isolation of the fluorescent moieties, we hope to establish conditions t,hat will permit us to diatinguish between natura,lly occurring and artif act’ually produced compounds. MATERIALS

AXD

METHODS

Matetiall. The following compounds were used as purchased from Mann Research Laboratories : glycine, alanin?: valine, leucine, isoleucine, glutamic acid, aspartie acid, threoninc i&o), phenylalanine, tryptophan, ‘Supported by the Medical Research Council of Canada., the American Heart and the Canadian Arthritis and Association, the Manitoba Heart Association. Rheumatism Society. ’ Visiting Scientist of the Medica. Research Council of Canada. Permanent address : Department of Biochemistry, Faculty of Medicine, Charles University, Hradec Kralove, Czechoslovakia. 3 Medical Research Associate, Medical R,esearch Cmnail of Cans&. 174

FLUORESCEXT

SUBSTANCES IN PRCWEIKS. I

175

proline, cystine, methionine, lysine, arginine, all as Dr.,-isomers, and L-cysteine. L-tyrosine was obtained from Nutritional Biochemical Corporation, and L-aspa,ragine, I;-glutamine, L-cysteic acid, and m-kynurenine sulfate from Sigma Chemical Corporation; L-hi&dine from British Drug Houses Ltd., and 3-chlorotyrosine from K R; Ii Laboratories, Inc. Dityrosine was prepared in our laboratory from L-tyrosine, hydrogen peroxide, and horseradish peroxidase (1). T’reahnent of Amino Acids with HCZ. Each of the amino acids was subjected to a procedure analogous to that of acid hydrolysis of proteins. The amino acid was heated with a 3Wfold excess of 6N HCl (fluorometric grade, Hartman-Leddon Company, Philadelphia) under nitrogen in sealed ampoules at a 1lO’C for 54 hours. The cooled contents of the ampoules were transferred quantitatively through a funnel with a coarse fritted disc and HCl was removed by evaporation under reduced pressure at 42”. Gel Filtration of Acid “Hydrolyxates.” Each of t’he acid-trea#ted amino acid solutions was eluted with 0.5 M acetic acid from a 46 X 0.9 cm column of Bio-Gel P-Z (200-400 mesh, Bio-Rad Labora,tories). The effluent was monitored with an ISCO ultraviolet analyzer (model UA-2, dual-beam optica’ unit, 280 nm) and 3.0 m1 fractions were collected. Fluorescence Measuremen~ts. Fluorescence wa,s determined with an Aminco-Bowman spectrofluorometer. All spectra are uncorrected and the values are expressedin arbitrary units. Descending Chromatography. This carried out on What’man 1 paper using butanol/acet,ic acid/water (60: 15:25). Components were localized by noting fluorescenceunder ultraviolet light, by ninhydrin spray, and by a rragent believed to be specific for t.yrosine derivatives (2). RESULTS

of

Pure Amino Acids. Of the typical amino acids found in prot,eins, it is generally considered that only tryptophan and tyrosine, and Do a lesser extent phenyla’lanine, are fluorescent in the ultraviolet region (3). To confirm this point, we examined the fluorescence of 22 amino acids dissolved in dilute HCl, pH 1.75, at a concentration of 1.0 ,pmole/ml, The pH was c.arefuIly controlled and blank values were subtracted from the fluorescent readings. The acidified samples were measured at 3 different wavelengths (see Table I; the basis for this choice of wavelengths is given in the “Discussion”), the samples adjusted to pH ‘11.5by the addition of NaOB, and fluorescencemeasurements repeated at the same wavelengths. In a.ddition to the fluorescence emitted by t,he aromatic amino acids, significant fluorescence was seen in solutions of the sulfur-cont’aining amino acids, methionina and cystine, Fluorescence

“Nonhydrolyxed”

176

LEDVlNA

AND

LABELLA

A 305 Ii’ 445

Amino acid

Acid

Cystine Methionine Phenylalanine Tyrosine Tryptophana

.I 275 P 305 --.-1__ Arid Alk.

-.:Uk.

1.3;s 1.4 10

1.2 211 9

5.7 170

1.4 4.9 6

.1 3’20 F 405

I- -... .-..II ..-Acid Alk. I .x0 0.86 1.46 2.2 2 9

x3 56

F’ maxima _-__.______ Acid

Alk.

360 360 288 305 352

340, 410 362, 412 288 408 362

U The fluorescence at optimal wavelengths in acid at 290-35‘2 nm is 479 and in alkali at 297362 nm it is 1919. Amino acids not listed showed negligible or no fhlorescence. A = act,ivation, P = fluorescence. Ail wavelengths in nm (uncorrected). Concentration = 1.O bmole/ml. An&co-Bowman spect’rofluoromet,er. Values transferred to meter multiplier 0.01. For pH, see text,. Absence of figures indicates absence of ~~IWWcence.

and also of arginine. Other amino acid solutions exhibited low fluorescence, which could be due to the presence of contaminants. Fluorescence of “Acid-Hydrolyzed” Pure Amino Acids. Each of the pure amino acids was exposedto conditions of acid hydrolysis of proteins (see “Methods”). “Hydrolyzed” samples were evaporated, redissolved in water to a concentration of 1.0 pmole/ml, and the pH a,djusted to 2.7 with HCI. Fluorescence was measured at. pH 1.75 and 11.5. The results are shown in Table 2. Even after treatment with HCI, fluorescence of most of the amino acids remained at a very low level, although the Fluorescence of “Acid Hydrolyzed”

A 305 F445 Amino acid Isoleucine Serine Cysteine Cysteic acid Methionine Histidine Arginine Phenylalanine Tyrosine Tryptophan Conditions

Acid

Alk.

ii 275 P305 Acid

A 320 P 405

Alk.

1.2 1.18 1.02 1.01 1.34 9.7

7.2 394

0.w 1.26

17 395

Amino Acids

1 .Y6 cl.!)1 1.82 1 .5fi 2.30 2.68 1.77

0.80 3 332 5.6

Acid

F maxima Alk.

1.05

1.91 1.38 1.01

2.9

correspond to those on Table 1.

5.4 1235

100 1276

Acid

Alk.

360,390 360,412 390,357 357,390 360,390 445 355 28% 305 3x7

360,400 360,400

358,396 357,400 356,400 288,330 410 38X

I-ulues were in gcircnbl slightly high(lr I I~:LIIthoS;ci0C Iirrl.rtAatedSolutionti. Significant increases in Auort:ticc-!llct~ orv~1~1~~~~1 \vii 11~cvcral of tJ~(: “hydrolyzed” amino acids. 1111 t,hc C:W of l)iatitlinc, a ww suh~tancc wit’h P maximum at 445 nm a,ppearetl which VW not found in t.he other acidtreated amino acids. With t8yrosincNUN quantitative change in fluorcscence at acid pH occurred, and at pH 11.5 the emission maximum shifted to 409 nm. The most striking effect, was seen for tjryptophan. Many new fluorescent substances were formed by HCl treatmt~nt, t,he most significant product being tha,t with A/F 320405 nm. Fluorescence

Spectra

of Amino

Acids

before and after “Acid

Hy-

droEysis.” Complete fluorescencespect’rn of tyrosine and tryptophan were carried out on solutions before and after treat,ment with HCl. The spectrum for trypt,ophan, but not tyrosine, was altered after treatment with HCl; a double activation peak with maxima at 292 and 320 nm developed on treatment’ of tryptophan with HCI (Fig. 1). The emission

2345678910

4

500 400 Wavelength hn)

600

700

FIG. 1. A/F spectra of tryptophan before (A) and after (B) acid “hydrolysis.” Solvent, HCI, pH 2.66. Concentration, 0.25 ,umole/ml. Meter multiplier 0.1. Dashed line, activation spectrum (fluorescence at 355 nm) ; solid line, fluorescence spectrum (activation at 288 nm). Insert C: Effect of pH on fluorescence of “nonhydrolyzed” tryptophan measured immediately (0) and measured 24 hours later (X). A/F 288-355 nm.

spectrum was shifted from 350 to 385 nm. Histidine fluorescence, which before acid treatment was negligible, was considerably increased after HCl treatment, although still very low compared to that of tryptophan and tyrosine (Fig. 2) . Gel Nh-attin of “Acid-Hydrolyzed” Amino Acids. “Acid-hydrolyzed”

178

LEDVINA

200

300

AND

LABELLA

500

400 Wavelength

600

(nm)

Fza. 2. A/F

spectra of acid “hydrolysis” of L-histidine. Concentration,0.57 pmole/ml (as base). Dashed lines, activation spectra (fluorescence at 445 nm) ; solid lines, fluorescence spectra (activation at 305 nm). Upper curves, in 0.5 M acetic acid ; lower curves, pH 12.0. Meter multiplier 0.001.

fluorescent components produced from trypt’ophan or tyrosine were separated on Bio-Gel P-2 (Figs. 3 and 4). The products produced by acid treatment of tryptophan exhibited strong fluorescence especially at A/P 345380 nm. The fluorescence of products resulting from acid treat’ment, like tryptophan itself, were relatively independent of pH. On the other hand, the elution diagram of the tyrosine “hydrolyzate” A

1.0 280

.5 : 80 70.

>

5

60

5 r

50.

g 5> L

1

A

40. 30. 20-

Fraction

Number

FIG. 3. Elution diagram on &o-Gel P-2 of “acid-hydrolyzed” DL-tryptophan. Elution wss performed with 0.5 M acetic acid and 3.0 ml fractions were collected. (A) fluorescence and W absorbance in 0.5 hf acetic acid. (EL) pH 11.50. Solid lines, A/F 3OSS45 nm; dotted lines, A/F 285-360 nm; dashed lines, A/F 345-380 nm. Meter multiplier set to 0.1. B-21 exhibits an F maximum at 410 nm.

FLUORESCENT

SUBSTBNCES

IN

PROTEINS+

I

179

::;: . . .I.. :: : “: : ::: ::” . . :3:

loo-

90-

::: .-: ::. :: : : :

80-

.7 A

280

.6 .5 .4

10 Lr I.

r+ 5

... 15

25

.,.. 35

0 305-44s 45

275-305 J 5~5

Fraction Number

FIG. 4. Elution diagram on Eo-Gel P-2 of “acid-hydrolyzed” L-tyrosine. Values represent A/F in nm. Meter multiplier set to 0.03. Other conditions identical with thvse on Figure 3. B-19 has A/F at 320405 nm.

showed a double fluorescencepeak at acid pH, presumably reflecting unaltered tyrosine together with its oxidation product, and corresponded to the UV absorption values of the effluent. A small additional fluorescence peak occurred around fraction 35 but its fluorescence did not appear to be that of typical phenols. At pH 11.5 the fluorescence of tyrosine was practically eliminated, as reported also by Cornog and Adams (4)) and a substance with A./F of 305445 was detected near fraction 19. Paper Chro~matogra.p,hy of Products of Tryptophan

and Tyrosine “Hy-

drolysis.” Several tryptophan breakdown products, resulting from treatment with HC1 under conditions simulating acid hydrolysis, were demonskated by paper chromatography. At least 4 fluorescent spots were detected, one of which had a fluorescence spectrum similar to that of kynurenine. This latter component, as well as one other, was ninhydrinpositive. There was no ninhydrin or fluorescent spot corresponding to tryptophan, indicating that the amino acid was completely destroyed by acid “hydrolysis.” On the other hand, the tyrosine “hydrolyzate” showed only one fluorescent ninhydrin-positive spot which corresponded to the

tly.rosine standard. Two partially rctsolved con~pc~n~:~~ t s obtained by gt!I filtration of the t,yrosine “hydrolyz:tle” (see above) wtlre appa~~tly r10t separated on paper, It should be pointed out that absenceof degradation products occurred onIy with soIutiolls of pure tyrosine and that fluorescent, products did in fact result from acid t,reatment of more complex tyrosincbcontaining compounds ( 14j .

The present observations show that conditions of acid hydrolysis promote the production of fluorescent substances from pure amino acids. It remains to be determined if these findings can be related to hydrolyzates of proteins and peptides. There are several possible complications with protein hydrolyzates: first, quenching of fluorescence by ot’her amino acids may occur, e.g., the strong quenching of phenols (5) and tryptophan residues (6) by carboxylic acids (7), or by other more complicated interactions (see survey by Cowgill (8, 9) ) . An intermolecular transfer of the excited state from the phenyl to the indole group { 10) can aIso apparently account for fluorescencequenching. Second,fluorescent substances which may be an integral part of the protein structure may b,e released by acid hydrolysis, e.g., fluorescent aldehydes ancl biphenyl have been found in collagen, elastin, and ot’her structural proteins (1, 11, 12). Of course, a preliminary chromatographic separation of the hydrolyzate might possibly resolve the naturally occurring from the artifactually produced fluorescent substances. Such a procedure may in fact be necessary in attempts to identify fluorescent compounds occurring naturally in certain structural proteins. In the present experiments, rigorous attempt’s were made to prevent oxidation of samples during “hydrolysis.” Although the vials were sealed under nitrogen, t’races of atmospheric oxygen may still be present, and we hope to examine the effects of added reducing agents. In strong alkali, i.e., pH values above the pK 10.1 of tyrosine, the fluorescencemaximum shifted to 345 nm at low concentrations of tyrosine and to 400 nm at higher concentrations (4)) the latter value agreeing wit,h our finding of 409 nm and of 400 nm by Longin (13). The fluorescence wavelengths at’ which the pure amino acids were examined include A/F 275-305 nm, corresponding to the tyrosine maxima, and 32&405 nm for dityrosine (,l, 11) ; however, indole derivatives also fluoresce intensely at wavelengths 320-405 nm. The values of 305-445 nm correspond to the maxima of major fluorescent substances found in acid hydrolyzates of proteins in general. The intenseIy ffuorescent substances produced by acid “hydrolysis” of tryptophan suggest t’heir possible use for the analysis of this amino acid,

FIJJORESCENT

SUBST~C~

IN

PROTEINS.

181

I

thus eliminating the necessity for its calorimetric determination or a separate alkaline hydrolysis. SUMMARY

Fluorescence of solutions of pure amino acids and their degradation products produced by conditions simulating acid hydrolysis of proteins was studied. Fluorescence data at three different wavelengths are presented for 22 amino acids at acid and alkaline pH, both before and after treatment with 6 N HCl. Acid treatment increased the fluorescence intensity of solutions of phenylalanine or histidine slightly, more so for tyrosine, and very markedly for tryptophan. Gel filtration of “acidhydrolyzed” amino acids revealed several newly formed fluorescent compounds from tryptophan and one from tyrosine. REFERENCES 1. K~LEY, F., LABELLA, F., AND QUEEN, G., Biochem. Biophgs. Res. 156 (1969). 2. ACHER, R., AND CROCKER, C., Biochim. Biophys. Acta 9, 704 (1952). 3. UDENFRIEND, S., “Fluorescence Assay in Biology and Medicine,” Academic Press, New Yo’rk/London, 1964. 4. CORNOG, J. L., JR., AND ADAMS, W. R., Biochim. Bio$ays. Acta 66, 5, WOLD, F., AND WEBER, G., Federation Proc. 22,348 (1963) _ 6. EDELHOCH, R., BRAND, L., AND WILCHEK, M., Biuchmtitry 6, 547 7. Cow~m, R. W., &whim. Biophys. Acta 100, 36 (1963). 8. COWGILL, R. W., Biochim. Biophys. Acta 168, 417 (1968). 9. CGWGILL, R. W., B&him. Biophys. Acta 168, 431 (1968). 10. WEBE&, G., Btichem. .I. 75, 33!j (1960). 11. L.&ELLA, F., KEELEY, F., VIVIAN, S., AND THORNHILL, D., Biochem. Commun. 26, 748 (1967). 12. ANDWSEN, S. O., Acta Physiol, Stand. 66, Suppl. 263 (1966). 13. JANGIN, P., Compt. Rend. 248, 1971 (1959). 14. Unpublished observations.

Commun,

34,

pp. 125-190. 356 (1963). (1967).

Biophys.

Res.