Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes

ANALYTICAL

BIOCHEMISTRY

95, 228-235

Intramolecularly

(1979)

Quenched Hydrolytic

A. YARON,~ A. CARMEL, Department

of Biophysics,

Fluorogenic Enzyme9

Substrates

for

AND E. KATCHALSKI-KATZIR

The Weizmann

Institute

of Science,

Rehovot,

Israel

Received November 7, 1978 The design and application of a recently developed type of fluorogenic substrates for proteolytic enzymes is reviewed. The substrates consist of peptide chains constructed to match the specificity of the particular enzyme and to bear a suitable chromophore at each side of the cleavable bond. One of the chromophores is a fluorescent group and the other is a quencher that causes a great reduction of fluorescence intensity of the fluorophore, either by direct intramolecular encounter or by radiationless resonance energy transfer. Enzymic cleavage of the molecule is followed by release of fluorescence as the result of cancelling the quenching interaction between the chromophores. The properties of such substrates and their possible future applications are discussed.

The preparation of a highly specific substrate for a given proteolytic enzyme usually requires the synthesis of specific oligopeptide of sufficient length to interact with as many subsites of the particular active site as possible. The hydrolysis of such an oligopeptide can be followed by a number of methods such as calorimetry, potentiometry, or spectroscopy. The latter method is usually preferred because of high sensitivity and convenience. Spectroscopic monitoring of proteolytic activity is possible only if a change in absorbance occurs during the cleavage of a specific bond linking a suitable chromophore to the rest of the molecule. A variety of chromophore-bearing oligopeptide derivatives have been successfully developed and utilized in assaying proteolytic enzymes. The most sensitive spectroscopic assay makes use of fluorescent groups as chromophores. Among such substrates it is worth

mentioning the napthalene and coumarine derivatives of amino acids or peptides which have been employed in the assays of aminopeptidase (1,2), carboxypeptidase (3,4), trypsin, chymotrypsin, and elastase (5,6). The intact substrates are only poorly fluorescent, whereas the free fluorophores, which are liberated by enzymic hydrolysis, are highly fluorescent. In the above spectrophotometric methods the fluorophore has to be linked directly to the bond undergoing enzymic cleavage. Most of the fluorophores used are bulky aromatic groups, different in nature from the amino acid residues that usually occur in natural substrates. The necessity of placing such bulky groups directly at the point of catalytic cleavage, as in the case in the above mentioned fluorogenic substrates, often represents a marked drawback. Also, the bond of the synthetic derivative differs from that joining the amino acid residues of the corresponding native substrates. A relatively recent development in construction of fluorogenic substrates follows an approach that allows the preparation of derivatives with an uninterrupted peptide

1 This paper is dedicated to the memory of Dr. Alvin Nason. 2 This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. 0003-2697/79/070228-08$02.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

228

FLUOROGENIC

chain. A fluorescent group is attached to one end of the molecule and another group, that can quench the fluorescence, is linked to the other end. The interaction between the two groups is quite efficient even when they are separated by several amino acid residues. Cleavage of the peptide chain at any point between the interacting groups results in separation between them, causing the appearance of fluorescence which is an accurate and sensitive measure of the number of substrate molecules cleaved. In the following we will deal with two types of intramolecularly quenched fluorogenie substrates, according to the nature of the quenching interaction: (1) quenching through collision between the fluorophore and the quencher and (2) through nonradiative electronic excitation energy transfer between a fluorescent donor and a suitable acceptor. Fluorogenic substrates quenched by intrunmlecular collision. The spectral properties of a fluorescent molecule can be affected by interaction with the environment in which the fluorophore is placed. An interaction with another molecule may change the fluorescence intensity by a number of mechanisms. Static quenching occurs when a fluorescent group forms a “dark complex” by interaction with a quencher prior to excitation. In dynamic quenching, the encounter with a quencher during the fluorescence lifetime competes with emission for depopulation of the excited state. In contrast to the long-range quenching by “resonance energy transfer” acting at a distance of approximately lo- A (to be dealt with in the next section), dynamic and static quenching requires actual short-range interaction and we will refer to them as “collisional” or “contact” quenching. Static and dynamic quenching has been theoretically treated by a number of authors. A general treatment of quenching by collision and complex formation was developed by Vaughan and Weber from considerations of the rate equations involved [Ref. (7) and

229

SUBSTRATES

references therein]. was assumed: -

A’

+ Q

The following

s k-’

Id: t. IA t I + A+Q

scheme

A’Q I A I

; I 1 &

AQ

In this scheme the potentially fluorescent molecule A was assumed to interact with the quencher Q to form the “dark” complex AQ in a reaction characterized by an equilibrium constant K,,. Encounter between A* (excited A) and Q leads to formation of the excited complex A*Q. The rates of absorption transition are Eand E’ for A and AQ, respectively. The rate of radiationless deactivation of the excited molecules A* is E.C,and A is the rate of emission. The primed quantities p’ and A’ refer to the respective values of the complex A*Q. The rates of association of A* with Q and dissociation of A*Q are k+* and k-*, respectively. Quenching interactions of the above type have been investigated in many systems that found useful application in various areas, e.g., in analytical chemistry (8), in the study of the exposure of membrane proteins (9), and in the investigation of liposome-cell interactions (10). When a quencher is incorporated into a molecule bearing a fluorescent group, and if interaction between these groups is structurally possible, then a short distance between the fluorophore and the quencher insures a relatively high frequency of collisions leading to effective depression of the fluorescence. At low concentrations cleavage at any point between the two groups renders the quencher ineffective due to the high dilution. The design of intramolecularly quenched fluorogenic substrates for hydrolytic enzymes, and particularly for peptidases, has to take into consideration the following: The peptide bond to be cleaved by the enzyme

230

YARON,

CARMEL,

AND KATCHALSKEKATZIR

. Wavelength

’ 410

’ 430

’ 450

’ 470

;-

-0

490

(nm)

FIG. 1. Absorption spectra of ABz-Gly (-), ABz-Gly-Phe(NO.&Pro (-.-.-), and Phe (NOz)-Leu (- - -), and the emission spectrum of ABz-glycine (. . .). The compounds (0.1 mM) were dissolved in 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl [the Fig. is taken from ref. (15)].

should be flanked by amino acid residues that provide good interaction with the enzyme active site; the fluorophore should be small sized in order not to interfere with the substrate-enzyme interaction, the quencher should have low absorption at the wave length suitable for excitation, and finally, the whole molecule must be resistant to other enzymes and sufficiently hydrophilic to be soluble in water. A few fluorogenic substrates for proteolytic enzymes such as leucine aminopeptidase (1 l), aminopeptidase P (12), elastase (13,14), and angiotensin-l-converting enzyme (1% 17) have been recently prepared and investigated (14). The substrate o-aminobenzoylglycyl-p-nitro-rphenylalanyl-L-proline [ABz-Gly-Phe(NO,)Pro],3 (I), for the assay of peptidyl-dipeptide hydrolase known as angiotensin- I-converting enzyme (EC 3.4.15.1) in mammals and as “dipeptidyl carboxypeptidase” in bacNH-CH2-CO-NH-CH-CO-N-CH-COOH d0

I Cb

U

&HZ @ NO*

teria (18) may serve as an example. The absorption and emission spectra of this substrate are shown in Fig. 1. Specificity of this enzyme requires the presence of a free 3 Abbreviations used: AB,-Gly-Phe(NO&Pro, aminobenzoylglycyl-p-nitro-~-phenylalanyl-~-proline.

o-

carboxyl group in a peptide molecule consisting of at least four amino acid residues or of an N-acyl tripeptide in which the penultimate C-terminal residue is not a proline. The N-acyl tripeptide ABz-Gly-Phe(NO,)Pro fulfills these requirements. Moreover, this structure is resistant to most carboxypeptidase and chymotrypsin activities because of the C-terminal proline and to aminopeptidases due to the absence of an a-amine. The o-aminobenzoyl group (ABz) is a well known small-size fluorophore with a high quantum yield. The p-nitrophenylalanine was selected since nitroaromatic compounds are well-known quenchers which participate in numerous charge-transfer complexes as electron acceptors. Although the kind of quenching interaction has not been so far elucidated, the long-range resonance energy-transfer mechanism (19) is not operating, since a spectral overlap between the absorption band of the quencher and the emission spectrum of the fluorophore is absent. Preliminary nmr studies (20) of ABzGly-Phe(NO,)-Pro in methanol as compared to o-aminobenzoylglycine showed an upfield shift (0.7- 1.0 ppm) of the Abz ring protons caused by the ring current of the p-nitrophenylalanine group. This effect indicates a face-to-face orientation of the two aromatic rings and suggests a contact quenching mechanism.

FLUOROGENIC

SUBSTRATES

Although the fluorescence spectrum of ABz-Gly-Phe(NO,)-Pro and of the nonquenched ABz-Gly have the same pattern, the fluorescence intensity of the later compound is about seven times higher. Therefore, the enzymatic cleavage of the GlyPhe(N0,) peptide bond can be monitored by recording the increase in fluorescence intensity. The angiotensin- l-converting enzyme present in human and guinea pig serum and calf lung, as well as the bacteria1 enzyme from Escherichia cob, were found to hydrolyze the above fluorogenic substrate. Fluorescence measurements enabled the assay of these enzymes in a sensitive, specific, and accurate manner. The intramolecular fluorophore-quencher interaction depends on distance between the two groups and on the structural characteristics of the molecule. In a series of aminopeptidase substrates (2) where X stands for one or two amino acid residues, the fluoresH*N F”-

co

x

0 -

CH*

(2) x 7 one or IWO Ornl”O OCld rewdues

cent ABz group was attached to the e-amine of lysine and the quenching p-nitrobenzyl group linked in ester form to the a-carboxyl group (11). As in the above described substrate for converting enzyme, no overlap between the fluorescence spectrum of l ,N(o-aminobenzoyl)lysine and the absorption spectrum of the p-nitrobenzyl group was observed, therefore the possibility of quenching by the Forster mechanism of resonance energy transfer is excluded and the quenching has to be attributed to a collisional mechanism. The quenching efficiency is expected to fall off with the distance between the interacting groups because of the decreasing probability of intramolecular encounter. In this case the rise in fluorescence intensity accompanying the complete release of Lys/ABz) by leucine aminopeptidase changes from 27-fold to l&fold when

231

going from the dipeptide to the tripeptide, respectively. These data indicate that further increase in the distance between the fluorophore and the quencher may still yield useful fluorogenic substrates. In fact, it is possible that at certain chain length of longer peptides efficient quenching may occur as the result of the flexibility of the longer substrate molecules. Many fluorophores and quenchers can be chosen. For example, other fluorophores. in addition to the o-aminobenzoyl moiety, e.g., anthracene, tryptophan, and aminoethylnaphthylamine, were found by us to be quenched by nitroaromatic groups. The only spectral requirement is a low absorption by the quencher at the excitation wavelength of the fluorophore. This requirement stems from the limit imposed on measurements of fluorescence by the “inner filter effect:” namely, the optical density at the excitation wavelength should not be higher than 0.1. This limitation should be taken into account when measurements of enzymic activity are performed at relatively high substrate concentrations. In the case of ABz-Gly-Phe(NO,)-Pro, the absorption of the nitrophenyl group is quite high (9860 M-’ cm-‘) at A,,,,, = 284 nm. However, at a higher wavelength, where the absorption band of the n-aminobenzoyl group is located (A,,,, = 312 nm), it decreases to about 30% of its maximal value. Above 330 nm the absorption is less than 10%. One can thus excite the fluorophore at this wavelength without marked interference by the absorption of the quencher. Measurements at very low enzyme concentrations require a high fluorescence yield of the fluorophore. efficient quenching, and a high turnover number for the substrate. With ABz-Gly-Phe(NO,)-Pro as substrate, rates of 0.03 nmol/min were conveniently measured for the converting enzyme. If the substrate should be applicable for assays of activity in a crude enzyme source, the excitation wavelength should not coin-

232

YARON,

CARMEL,

AND

tide with the main absorption band of the contaminating proteins. The fluorophorequencher pair ABz and Phe(N0,) is not affected by the absorption band of proteins centered at 280 nm and is suitable for constructing substrates applicable to activity measurements in native media such as human serum. Thus, the low levels of converting enzyme present in human serum can be reliably measured (17) at favorable substrate concentrations (5 times Km) of ABz-Gly-Phe(NO,)-Pro. The advantage of intramolecularly quenched substrates, that they do not require direct participation of the chromophores in the cleavable link is demonstrated by the recently developed substrate of aminopeptidase P (EC 3.4.11.9). Specificity of this enzyme requires that the N-terminal amino acid of an oligopeptidase substrate is followed by a proline residue. Therefore, amino acid naphthylamides, commonly used as fluorogenic substrates for aminopeptidases, are not applicable in this case. On the other hand, the intramolecularly quenched proline-containing tripeptide Phe(NO,)-ProLys-(ABz) has been found (12) suitable for the fluorimetric assay of aminopeptidase P. Cleavage of the Phe (NO,)-Pro bond results in the greater fluorescence of the aminobenzoyl residue. Fluorogenic nance energy

substrates transfer.

quenched by reso-

Resonance energy transfer, known also as nonradiative energy transfer, is a mechanism in which an excited fluorophore-the donor-transfers its excitation energy to another chromophorethe acceptor. This transfer is not mediated by direct contact of the chromophores and results in the quenching of the fluorescence of the donor. If the acceptor is fluorescent too, excitation of the donor at wavelengths where the acceptor is not excited directly leads to an emission characteristic of the acceptor. The theory of the resonance energy transfer was formulated by Forster (19) who

KATCHALSKI-KATZIR

expressed the efficiency,

e, of the transfer

by Eq. [cl e

=

l-

F

Fo

-

ro6

,

r6 + ro6

EC1

where F and F. are the fluorescence intensities of the donor in presence and absence of the acceptor, respectively, r is the distance between the centers of the two chromophores, and ro, which equals the distance between the donor and the acceptor when the efficiency is 50%, is given by Eq. [d] r. = (8.78

X

1O-25 K2qJ/n4Y

IdI

where ~~ is an orientation factor, q is the donor quantum yield in the absence of transfer, J is the spectral overlap intergral, and n is the refractive index of the solvent. The main consequence of the theory is that the energy-transfer efficiency depends directly on the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, and decreases with the sixth power of the distance between the chromophores. The latter dependence of energy transfer on distance raises the possibility that energy donor-acceptor pairs might be used to reveal proximity relationships in biological macromolecules. The theoretically predicted energy transfer-distance dependence was experimentally verified with synthetic models carrying suitable acceptor-donor pairs at a defined distance (21). Intramolecularly quenched fluorogenic peptides, containing an enzymatically cleavable bond situated between an energy transfer donor and acceptor, were investigated (22) and shown to be interesting fluorogenic substrates for proteolytic enzymes. Thus, the Lys-Ala peptide bond situated between the anthracene acceptor and the naphthalene

FLUOROGENIC

donor in anthracene-9-carbonyl-p-alanyllysylalanyl-Znaphthyl methylamide, (3) is cleavable by trypsin. The hydrolysis causes separation of the two parts of the molecule and interrupts the intramolecular quenching of the naphthalene donor mediated by the nonradiative energy-transfer mechanism operating in the intact molecule. Consequently, a high increase of fluorescence, characteristic of the naphthalene donor, is observed which is a measure of the extent of the trypsin-catalyzed hydrolysis. Simultaneously with the increase of the naphthalene fluorescence, emission of the anthracene acceptor excited by irradiation of the naphthalene moiety decreases; this change can also be used for monitoring the hydrolytic reaction. A series of analogous CO-PAla-Lysn-NH000 OS3

‘.X2

m

n=2.3.4

233

SUBSTRATES

(4)

substrates of the general formula (4), which have the same absorption spectra as (3), undergo tryptic scissions at different sites. Therefore, the rate of occurrence of the first enzymic cleavage cannot be determined readily by conventional methods. However, it is the scission of the first amide bond of the intact molecule which leads to a drop in the yield of energy transfer, regardless of the site of cleavage. Any additional hydrolysis of the initial degradation products will not affect the fluorescence efficiency of the naphthalene donor. By monitoring the increase in fluorescence of the naphthalene moiety at 340 nm upon excitation at 280 nm, the kinetic parameters K, and k,,, were determined for the trypsin-catalyzed hydrolysis of this series. Linear double-reciprocal plots obtained in the low substrate concentration range (3 X IO+ to 3 X IOP M) coincided with the data obtained at relatively high substrate concentrations (2 x 10-j to 8 x 10e5 M) by the pH-stat method. With

increasing chain length n = 2, 3, and 4, K, decreased (2.4, 1.5, and 0.27 x lo-” M, respectively) and the catalytic efficiency expressed by the proteolytic coefficient C (==kcatlKm) increased (1.20, 2.52, and 4.95 x 10e6 M-’ s-l, respectively) about two-fold in each step of elongation (23). S%-

Glyn

- Trp

n=l.2,3

(5)

N(CH3)2

A series of carboxypeptidase A substrates of the general formula (5) was prepared (24) in which the tryptophan fluorescence is efficiently quenched by the dansyl (Dns) group by means of nonradiative energy transfer. Carboxypeptidase A is known to cleave peptide bonds adjacent to C-terminal aromatic acids and the hydrolysis rates of the synthetic substrates are enhanced if the N-terminal amino group is blocked. Enzymatic cleavage of the Gly-Trp peptide bond terminates the quenching of tryptophan fluorescence by the dansyl group and as the result, fluorescence of the tryptophan increases. It was thus possible to use this series of fluorogenic substrates to develop a highly sensitive and convenient assay for carboxypeptidase A. Experimentally, it was found that on irradiation at 290 nm the substrates Dns-Gly,-Trp, n = 1, 2, and 3, emit light at 575 nm, which is characteristic of the fluorescence of the dansyl group, and practically no fluorescence at 350 nm, which is characteristic of tryptophan emission. Cleavage of the Gly-Trp bond reduced the dansyl fluorescence and enhanced more than loo-fold the emission of tryptophan. The quenching efficiency was found to depend on the peptide length. In the series n = 1,2, and 3, the tryptophan fluorescence is quenched about loo-, 20-, and 4.5fold, respectively. Kinetic measurements with Dns-Gly-Trp over a broad range of concentrations yielded a linear double-reciprocal plot and the re-

234

YARON,CARMEL,ANDKATCHALSKI-KATZIR

sulting constants were in good agreement with those obtained by other methods at higher substrate concentrations. The spectral characteristics of the tryptophan residues of carboxypeptidase A, the dansyl group, and the Co2+ ion of Co2+-carboxypeptidase A reveal that the dansyl group can serve both as an acceptor of the tryptophanyl excitation energy and as a donor to the cobalt atom in a tryptophandansyl-cobalt energy-relay system (25). The energy transfer between the enzyme tryptophanyl residues and the dansyl groups in the substrates employed (Dns-Gly,-Phe, n = 1,2,3,4andDns-Gly,-Trp,n = 1,2,3) enabled the observation of the formation and breakdown of enzyme-substrate complexes. Subsequent to binding of the substrate, transfer of energy of the bound dansyl group to the cobalt atom allowed calculation of the distance between these moieties, taking into consideration the dipole-dipole resonance transfer in the basic process. In the derivation of the kinetic parameters characterizing the above substrate-enzyme interactions, the stopped-flow fluorescence method was most useful. By this method the rates of formation and disappearance of the enzyme-substrate complex could be readily determined. CONCLUDING

REMARKS

At present, only a few fluorogenic substrates, either internally quenched through collision or by long-range electronic energy transfer, have been studied. The applicability of such substrates for the assay and characterization of proteolytic enzymes is, however, by now firmly established. Fluorogenic substrates of high specificity can be readily designed and used for the determination of the corresponding enzymes in assays of high sensitivity. Moreover, when the spectral properties are well chosen, corresponding fluorogenic substrates can be used to determine directly specific enzymic activities

in biological complex systems, such as the serum or crude tissue extracts as well as in native systems such as intact cells and its components. If the fluorescent group liberated after hydrolysis of the fluorogenic substrate is precipitated or locally adsorbed, it should be possible to localize the proteases under consideration within cells and tissues. It should also be possible to study the permeability of the quenched fluorogenic substrates into native and artificial vesicles if the latter contain the corresponding hydrolytic enzyme. The principle outlined for the preparation of suitable fluorogenic substrates for proteolytic enzymes might be readily applied to other hydrolases, such as enzymes hydrolyzing oligosaccharides and oligonucleotides. An interesting intramolecularly contactquenched substrate, nicotinamide 1,N6-ethenoadenine dinucleotide, a fluorescent analog of the coenzyme nicotinamide adenine dinucleotide, has been synthesized and shown (26) to be hydrolyzed by Neurosporu crassa NADase (EC 3.2.2.6) and by Crotalus adamanteus venom phosphodiesterase (EC 3.1.4.1). In this substrate the ethenoadenine ring serves as the fluorogen and nicotinamide serves as the quencher. As a matter of fact, there is no appreciable overlap between the emission spectrum of the 1,N6-ethenoadenine moiety and the absorption spectrum of the nicotinamide quencher. Upon hydrolysis of the substrate nicotinamide 1,W-ethenoadenine dinucleotide, the fluorescence of the fluorophore increases IO-fold, due to the separation of the nicotinamide and the ethenoadenine rings. It is pertinent to note that the cancelling of the intramolecular-quenching interaction does not require, as a rule, rupture of the substrate molecule. An increase in distance between the quencher and the fluorophore, occurring as a result of substrate (or inhibitor)-enzyme interaction not leading to hydrolysis, might also cause cancellation of the quenching effect.

FLUOROGENIC

When comparing the two types of intramolecular-quenching mechanisms discussed above, one should bear in mind that contact quenching, in contradistinction to resonance quenching, occurs only if the substrate attains the adequate conformation that permits an encounter between the chromophores. On the other hand, quenching by collision does not require the specific spectral characteristics enabling nonradiative resonance energy transfer. A broader range of chromophores should therefore be available for the design of specific substrates, possessing fluorophores quenched by the contact mechanism. Finally, it is worth mentioning that excitation of the fluorescent moiety of fluorogenic substrates or inhibitors with polarized light and measurement by adequate equilibrium and kinetic methods (27) of the extent of fluorescence depolarization during the various stages of interaction with the corresponding enzyme might shed new light on the orientation and free rotation of the fluorogen in the enzyme-substrate or enzyme-inhibitor complexes, while interacting with the enzyme. It can be expected that the additional syntheses and studies of intramolecularly quenched substrates will be of considerable help in the study of the biological role of hydrolytic enzymes and in clarifying of their modes of action.

Zimmerman, M., Ashe, B.. Yurewicz, E. C.. and Pate], G. (1977) And/. Biochrnl. 78, 47-S I. Roth, M. (1963) C/in. Chim. Acta 8, 574-578. Vaughan, W. M., and Weber. G. ( 1970) Biochc~w istry

16. 982-986.

IO. Weinstein, J. N., Yoshikami, S.. Henkart. P.. Blumenthal, R.. and Hagins. W. A. (1977) .Sc,icnct 195, 489-492. 11. Carmel, A., Kessler, E., and Yaron, A. ( 1977) El/r. J. Biochem.

87, 265-213. 16.

Persson. A., and Wilson, 1. B. (1977) Attctl. Birt-

17.

Carmel. A., Ehrlich-Rogozinski, A., and Yaron. A. Clit7. Chirn. Actu. submitted for publication. Yaron. A. ( 1976) in Methods in Enzymology (Lorand, L.. ed.). Vol. 45. pp. 599-610. Academic Press. New York. Forster. Th. (1948) Anr7. Pl7y.t. 2, 55. Sheinblatt. M.. Carmel. A., and Yaron. A.. in preparation. Steinberg, I. Z. (1971) At7n. ROT. Eidrern. 40. 83-114. Carmel. A., Zur. M.. Yaron. A., and Katchalski. E. ( 1973) FEBS Lett. 30, 1I- 14. Zur. M. (1975) Ph.D. Thesis, Feinberg Graduate School, The Weizmann Institute of Science, Rehovot, Israel. Latt, S. A., Auld. D. S.. and Vallee. B. L. ( 1972)

chctn.

18.

19. 20. 21. 22.

24.

L. J. (1962) Biochem. Bioph~,~. Rc,,s.

9, 430-435. ( 1965) in Enzymes

2. Roth, M. in Clinical Chemistry (Ruyssen, R., and Vandenriesche, E. L.. eds.), p. 10, Eisevier, Amsterdam. 3. Ravin. H. A., and Seligman, A. M. (1951) J. B;o/. Chem. 190, 391-402. 4. Lasser. N., and Feitelson. J. (1971) Biochrrrzistr> 10.307-311.

73, 617-625.

12. Carmel. A., and Yaron, A., in preparation. 13. Atlas, D. (1974) Ph.D. Thesis, Feinberg Graduate School, The Weizmann Institute of Science. Rehovot. Israel. 14. Carmel, A. (1975) in Peptides: Proceedings of the Thirteenth European Peptide Symposium, lsrael, April, 1974. (Wolman. Y ., ed.). pp. 385391, Halsted Press, New York. 15. Carmel, A.. and Yaron. A. (1978) Eur. ./. Lliochem.

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SUBSTRATES

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For general discussion of these methods see for example in Chen, R. F., and Edelhoch, H. ( 1975) Biochemical Fluorescence: Concepts. Vol. I. Dekker. New York.