Evidence for protein self-association induced by excluded volume Myoglobin in the presence of globular proteins

316

Biochimica et Biophysica Acta, 670 (1981) 316-322

Elsevier/North-Holland Biomedical Press BBA 38743

EVIDENCE FOR PROTEIN SELF-ASSOCIATION INDUCED BY EXCLUDED VOLUME MYOGLOBIN IN THE PRESENCE OF GLOBULAR PROTEINS

JACOB WILF and ALLEN P. MINTON Laboratory of Biochemical Pharmacology, National Institute o f Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes o f Health, Bethesda, MD 20205 (U.S~4.)

(Received February 24th, 1981)

Key words: Hemoglobin; Myoglobin; Protein self-association; Fluorescence polarization; Excluded volume

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, dbonuclease A,/~dactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sup fonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

Introduction Analysis of the contribution of excluded volume to the thermodynamic activity of proteins in solution has led to a recent prediction [1] that protein selfassociation may be greatly enhanced in solutions containing high concentrations of unrelated protein species, which serve primarily to occupy a significant fraction of total solution volume. It was felt by the present authors that myoglobin, which normally exists as a monomer of molecular weight 17 000 in dilute solution, would provide a reasonable protein species with which to test the above prediction. The tertiary structure of myoglobin closely resembles that of the r-chain of hemoglobin, which, when separated from the a-chain of hemoglobin, forms stable tetramers in dilute solution [2]. It was reasoned that Abbreviations: Hb, hemoglobin; Mb, myoglobin; ANS, 1-anilino-9-naphthalene sulfonate; apoHb, apohemoglobin; apoMb, apomyoglobin; ANS-Hb, ANS-hemoglobin; ANS-Mb, ANS-myoglobin;PEG, poly(ethylene glycol).

perhaps myoglobin, like the /3-chain, possesses the potential to form well-defined oligomers, but with a rather more positive free energy of formation, so that under ordinary laboratory conditions such oligomers constitute a negligible fraction of total myoglobin. If such is the case, and if addition of unrelated spacefilling proteins greatly enhances protein self-association as predicted by theory [ 1], then in the presence of high concentrations of unrelated proteins a significant fraction of myoglobin may exist in oligomeric form. We here report the results of experiments designed to explore this possibility. Fluorescent derivatives of myoglobin and hemoglobin (called ANS,-myoglobin and ANS-hemoglobin, respectively) were prepared. ANS-Mb and ANS-Hb are known to exist primarily as monomers and dimers, respectively, in dilute solution [3,4]. The fluorescence polarization (a measure of the rotational relaxation time) of these derivatives was measured as a function of the concentration of added protein and poly(ethylene glycol)s. The results indicate that the state of association of ANS-Hb is

317 unaltered and that of ANS-Mb is altered upon addition of high concentrations of unrelated proteins. The simplest interpretation of the data for ANS-Mb is that in the presence of high concentrations of unrelated proteins ANS-Mb self-associates to form dimers which resemble ANS-Hb in overall size and shape. Materials and Methods Twice crystallized human hemoglobin (Hb), crystallized lyophflized equine skeletal muscle myoglobin (Mb), three times crystallized egg white lysozyme, three times crystallized lyophilized bovine ~-lactoglobulin, and five times crystallized bovine pancreatic ribonuclease A were obtained from Sigma and used without further purification. PEG-6000 (Carbowax, Union Carbide) and PEG-20 000 (Hoechst), were gifts from Dr. D. Atha of The Blood Research Laboratory, American Red Cross, Bethesda, MD. The three times crystallized magnesium salt of ANS was a gift from Dr. H. Edelhoch of the National Institutes of Health.

Preparation of fluorescent derivatives of Hb and Mb. ApoHb and apoMb were prepared by the following modification of the acid/acetone method [5]. An acid/acetone mixture was prepared by mixing 3 ml 1 M HC1 with 1 1 acetone. A cold 3% solution of protein was added dropwise to 30 vol. vigorously stirred acid/acetone mixture cooled to -20°C with dry ice. Stirring was continued for approx. 20 min after addition of all of the protein solution, and the precipitated apoprotein subsequently collected by centrifugation at 1 000 × g for 15 min at -20°C. The supernatant acetone was discarded and the colorless precipitate dissolved in the minimum amount of cold water necessary. The solution of apoprotein was dialyzed against 500 vol. distilled water for 6 h at 2°C to remove traces of acetone, and then dialyzed against 1.6 • 10 -3 M NaHCOa (pH 7.2 at 20°C) for 30 h at 2°C. During the course of the second dialysis a pink flocculate of dentaured protein appeared which was subsequently removed by filtration. The concentration of apoprotein in the clear, colorless ffdtrate was determined by ultraviolet absorption using the follcm lowing values of ezson m.. apoMb, 15.7 mM -I ; apoHb, 12.5 mM -1 . The efficiency of heme removal was quantitatively estimated from the ratio of the heights of absorbance peaks at 280 nm and 405 nm. According

to this assay, over 97% of the hemes were routinely removed from Mb and over 95% of the hemes routinely removed from Hb by the procedure described above. Stryer [3] showed that ANS forms a strong 1 : 1 complex with apoMb and a strong 4 : 1 complex with apoHb, presumably by occupying the vacant heme pockets. ANS-Hb and ANS-Mb were prepared by adding 10 -3 M ANS in increments of 2 pJ to a fluorescence cell containing 3.5 ml of the corresponding apoheme protein at a concentration of 10 -4 M binding sites, unless otherwise indicated. The solution was stirred after each addition of ANS and the fluorescence intensity was measured at an emission wavelength of 465 nm (see below). The addition of ANS was halted when the intensity reached a maximum. Using the binding constants given by Stryer [3], it was calculated that the fluorescence maximum correspond to a fractional binding site occupancy of 0 . 7 0.8.

Measurement of fluorescence and fluorescence polarization. Fluorescence spectra were obtained on a Perkin-Elmer MPF~4A spectrofluorometer equipped with Woods polarizing accessory and Polaroid polarizers. Spectra were recorded with excitation and emission slit widths of 1.8 cm (6 nm bandpass). All measurements were carried out at 20 -+2°C in phosphate (0.006 M) buffered physiological saline (0.15 M) at pH 7.2 or 7.4. Uncorrected excitation and emission spectra of the ANS proteins were essentially identical to those obtained by Stryer [3] at neutral pH and 20°C, with an excitation maximum at 375 nm and an emission maximum at 460 nm. For polarization measurements the excitation wavelength was fixed at 375 nm and emission spectra recorded between 400 nm and 550 nm for each of the four polarizer configurations (vv, vh, hv, hh). Intensity readings were taken from each of the four spectra at five wavelengths in the vicinity of the emission maximum (440, 450, 460, 470, 480 nm). The polarization was calculated for each wavelength using the relationship [5] : p - I w / h h - Ihv/vh

Iwlhh +Ihvlvh where I is the intensity in relative units. The reported value of p is the mean of the values calculated at the five wavelengths. No systematic variation o f p was ob-

318 o.~ beta-tactogtobultn

PE¢-6000 0.3~ O ~ I-,~ ¢e" 0.3 Z O I-(E O.~ r..J

0.3.' o I-(E ~ 0.3 Z .~r o I-~ 0.25 I',,t

~

~ m ~

ml~

~

~ ~ ~ ~1~1

~

(12 .-I

~

j

~

~m~ ~rq~

~

~ ~.

._1 O 0.~' A

o.1~

i

20

.

30

I

~

10

~'0

C Rddltive

(9/dl)

rtbonuc~ease

PEO-ZO000

30

(9/dr)

(1~.6K)

0.35 173 I-(K 1:~ 0.3 Z O I-(I:: O. 25 N

0.35

t~m

D

o.1~

i

10

CRddmtlve

~

~.

q~ ._1 O ~_ 0o?

._1

O O.Z .*,

E

B i

0.1.

O. 1~

i 20

10

C Rddlttve

tysozyme

(9/dr)

C

~

~

N

~ ~

~

.-ff ~

~

~ ~~

i

R d d l t #ve

20

30

(9/dr)

Fig. 1. Dependence of the polarization ratio of ANS-Hb (squares) and ANS-Mb (circles) upon the concentration of added polymer or protein. Upper dashed line calculated using Eqn. 4 with best-fit value of parameter B given below. Lower dashed line calculated using Eqn. 6 with same parameter value. (A) PEG,6 000, B = 0.021 dl/g; (B) PEG-20 000, B = 0.028 dl/g; (C) lysosyme, B = 0.056 dl/g; (D)/3-1actoglobulin, B = 0.026 dl/g; (E) ribonuclease A, B = 0.025 dl/g.

(13.9K1

O. 3.~ O I-. (E ~ 0.3 Z O ~ [] ~ I-(I: O.~'! N

i

10

30

0,4

~

~

_1 O I~_ 0.2 ¢ O. 1.'

~

~

~.

0 0.~' o_

O .1.,1~ ,",," 0.3 z O ~-(E 0.~'5 t.,,l

(18.4K)

i

10 C Rddtt tve

i

20 (9/dr)

30

served with wavelength in the region 440--480 nm, and scatter was usually about 3--4% of the mean value. Results of measurements performed in duplicate and triplicate indicate that the reported values of p are ordinarily reliable to within +0.005.

319 Results

ANS ÷ apoheme protein in the absence of macromolecular additive. The fluorescence polarization of ANS-Mb and ANS-Hb were determined to be 0.175 and 0.270, respectively, at 20°C in pH 7.2 or 7.4 phosphate-buffered saline, in reasonably good agreement with results obtained by Stryer under similar but not identical conditions [3]. These values were found to be independent, tO within experimental error, of the fractional occupancy of ANS binding sites over the range 0.1-0.7 in apoHb and 0.1-0.85 in apoMb. The polarization was also found to be independent of the concentration of apoMb or apoHb over the range I • 10 -~ M to 5 • 10-4 M binding sites, a range which corresponds to a several hundred-fold increase in fluorescence intensity. These findings indicate that the observed polarization (as opposed to intensity) in both ANS-Mb and ANS-Hb is an intrinsic property of the ANS-binding site complex only, and is not significantly dependent upon the concentration of occupied or unoccupied binding sites or upon the amount of free ANS in solution. Subsequent measurements were carried out on apoMb or apoHb at a concentration of 1" 10-4M binding sites which had been titrated with ANS to near-maximal fluorescence intensity, corresponding to a stoichiometric ratio of 0.7-0.8 ANS/binding site. ANS-proteins prepared in this manner were found to be stable at temperatures up to 30°C for periods of time much greater than those required to perform the experiments reported here. When solutions of ANS-Mb and ANS.Hb were cooled, the polarization ratio of both species increased monotonically to a maximum of 0.395 at the freezing point, when the solutions ceased to flow. ANS ÷ additive in thb absence of apoheme protein. The fluorescence intensity of 10-4 M ANS was measured in the presence of each of the macromolecular additives employed in the present study at several concentrations up to an including 25 g/dl. Under no conditions did the intensity exceed 2% of that observed in the same solution after the addition of 10 -4 M apoHb or apoMb. It follows that the fluorescence observed in solutions containing ANS, apoheme protein and macromolecular additive may essentially be attributed entirely to the ANS-apoheme protein complex: contributions from ANS-additive complexes are negligible.

ANS ÷ apoheme protein ÷ macromolecular additive. The fluorescence intensity of ANS-Mb and ANSHb solutions was observed to decrease upon addition of added polymer or protein. The extent of fractional decrease amounted to at most approx. 50% at the highest additive concentration employed in this study. It was observed that the fractional decrease in ANS-Mb fluorescence intensity at a given lysozyme concentration was the same, to within experimental error (-+10%), as the fractional decrease in ANS-Hb fluorescence intensity at the same lysozyme concentration for all concentrations up to and including 25 g/dl lysozyme. The polarization of ANS-Mb and ANS-Hb was measured as a function of concentration of added PEG-6 000, PEG-20 000, ribonuclease, ~-lactoglobulin and bovine serum albumin; the results are plotted in Figs. 1A-1E.

Discussion The objective of the present study is to discern whether any feature of the observed dependence of fluorescence polarization of ANS-Mb upon additive concentration may be attributed to self-association of ANS-Mb in the presence of additive. Since changes in fluorescence polarization may in general be attributed to changes in any of several variables, our analysis is based upon comparisons rather than absolute interpretations.

(1} Comparison of the effect of PEG and proteins upon fluorescence polarization of ANS-apoheme proteins Because poly(ethylene glycol) is characterized by an open random-coil conformation in solution, excluded volume effects arising from addition of PEG to a protein solution are thought to be substantially smaller than compact globular protein [1]. PEG is therefore employed as an additive which is not expected to induce self-association of ANS-Mb at the concentrations used in this study. This expectation is supported by the observation that the viscositycorrected sedimentation coefficient of dilute myoglobin in buffer is identical to that of dilute myoglobin in 30 g/dl PEG-6 000 (Minton, A.P., unpublished data).

320

(2) Comparison of the effects of a given additive upon the fluorescence polarization of ANS-Mb and ANS-Hb The results of fluorescence polarization studies [3,4] indicate that ANS-Hb, like apoHb [5], exists primarily as a dimer of molecular weight 33 000 in dilute solution. Since a given additive has approximately the same effect upon fluorescence lifetime in ANS-Mb and ANS-Hb (see 'Results' and 'Appendix'), differences in the effect of a given additive upon the polarization of ANS-Mb and ANS-Hb may be attributed to differences in the effect of that additive upon the rotational relaxation time, and hence size and shape, of the two species. Both ANS-Hb and ANS-Mb behave as spherical or quasispherical particles in solution [3,4]. The fluorescence polariztaion of both species may thus be written in the form of the Perrin equation [6] :

1 PM

1 (~_ _~)3~'M --

1

0

PM

+

--

OM

1 -

o

+

(1)

where the subscripts M and H refer to ANS-Mb and ANS-Hb, respectively; r is the fluorescence lifetime of the fluorophore; p is the rotational relaxation time of the quasispherical molecule; and p° is the fluorescence polarization of the fluorophore in the absence of rotational relxation. The rotational relaxation time of a rigid particle in solution may be generally represented as r~ /9 = ~-~ a (2) where r~ is the effective solution viscosity; k is Boltzmann's constant; T the absolute temperature; and Q a function of particle size and shape only [7,8]. In the appendix we derive the following relationship between PM and PH at the same temperature and concentration of the same macromolecular additive: ('~M-2.5)_267(

QH

/QM ~

(3)

"

where Q~(O) and Q~O) are the values of Q for ANS-

lib and ANS-Mb, respectively, in the absence of macromolecular additive. The experimentally observed dependence of PH upon c, the weight concentration of additive, may be well described by the empirical function 1 PH

- 2.5 + 1.24 exp(-Bc)

(4)

where B is an independently variable parameter, the value of which depends upon the nature of the additive, and which may be determined by least-squares fitting of Eqn. 4 to the data for a given additive species. The upper dashed line in Figs. 1A-1E is the dependence of Pr~ upon c calculated using Eqn. 4 with the best-fit values of B given in the figure captions. Combining Eqns. 3 and 4, we obtain 1 - 2 . 5 + 3 . 3 1 exp(--Bc)(?H~,,~/?M~,~] \(~H~.U.I/(~M ~U)] Pra

(5)

The ratios Q~Q~O) and Qr~t/Qr~(O),which reflect changes in the effective size and/or shape of ANS-Hb and ANS-Mb, respectively, may be functions of c. If we assume that

a~i/arl(O) = QM/QM(O) for all values of c, then Eqn. 5 simplifies to 1

- 2.5 + 3.31 exp(-Bc)

(6)

PM The lower dashed line in Figs. 1A-1E is the dependence of PM upon c calculated using Eqn. 6 with the best-fit values of B given in the figure captions. It may be seen that the measured values of PM are in good agreement with the calculated values for all concentrations of added PEG-6 000 and PEG-20 000, and for low cortcentrations of added lysozyme and ribonuclease. As the concentration of protein additives increases, the value of PM exceeds that calculated according to Eqn. 6 and, in the limit of high protein concentration, becomes equal, to within experimental error, to the value ofpr~ calculated according to Eqn. 4. Equating the right-hand sides of Eqns. 4 and 5, we obtain the result that at high concentrations of protein additive

QM[Q~v~(O) ~- 2.68 Qn]Qn(O)

321 for all three additives employed in this study. The major experimental findings of this study may therefore be summarized as follows: (i) at all concentrations of PEG-6000 and PEG-20000, and at low concentrations of the protein additives, QM[QM(0) ~- QH/QI-I(O) and (ii) at high concentrations of all three protein additives, QM/QM(O) ± 2.7 Q~QH(O). As mentioned above, we observed via sedimentation velocity experiments that the molecular weight of myoglobin appears to be unaltbred in the presence of high concentrations of PEG-6 000. We shall assume that ANS-Mb, like myoglobin, neither self-associates nor associates with PEG in the presence of high concentrations of PEG. It follows that for all concentrations of PEG, Q~QFI(O) " QM/QM(O)= 1 ; that is to say, ANS-Hb, like ANS-Mb, neither self-associates nor associates with PEG in the presence of high concentrations of PEG. Our next assumption is based upon the observation that the dependence of PH upon the concentration of PEG-6 000 and PEG-20 000. If ANS-Hb were self-associating or associating with either of these proteins to any significant extent, Q~t would be larger by at least a factor of 1.4 at high concentrations of ribonuclease, and by at least a factor of 2 at high concentrations of /~-lactoglobulin, than in the presence of high concentrations of PEG [7,8]. In order for such differences in QH not to be manifested as significant differences in the dependence of PH upon the concentrations of these different species, one would have to invoke a complex and altogether ad hoc compensation of the factors appearing on the right-hand side of Eqn. A2b (see Appendix). It is much more simple and straightforward to conclude that the similarity between the observed dependence of Prt upon the concentrations of these four additives reflects similar behavior of ANS-Hb in the presence of all four additives. Hence we shall assume that ANSHb neither self-associates nor associates with added protein at high concentrations of added ribonuclease A or /~-lactoglobutin. This assumption is consistent with our earlier finding that the thermodynamic and hydrodynamic behavior of hemoglobin in solutions of up to 30 g/dl may be quantitatively accounted for without invoking self-association of hemoglobin molecules at high protein concentration [9-11]. In the light of the assumptions stated above, our experimental findings may be interpreted as follows:

(i) The effective size and shape of ANS-Hb are not altered upon addition of any of the macromolecular additives employed in the present study, with the possible exception of lysozyme. (ii) The effective size and shape of ANS-Mb are not significantly altered upon addition of PEG6 000 or PEG-20 000 up to concentrations of 25 g/dl. (iii) The effective size and/or shape of ANS-Mb appears to be altered upon addition of sufficient concentrations of ribonuclease, /~-lactoglobulin, and lysozyme. This is reflected in an increase in the value of QM from QM(0) to 2.7 QM(0) in the pre~ence of/~-lactoglobulin and ribonuclease, and from QM(0) to 2.7 Q~(O)[QH/QH(O)] in the presence of lysozyme. The effective size and/or shape of ANS-Mb could be altered by association of ANS-Mb with added protein. However, our experimental data suggest that the fluorescent species present at high concentrations of all three added proteins resembles ANS-Hb, which exists predominantly as a dimer of myoglobin-like subunits, both in the absence of macromolecular additive [3,4] and in the presence of high concentrations of ribonuclease and/3-1actoglobulin (see above). The increase in Qra accompanying the hypothetical self-association of ANS-Mb to form dimers may be estimated as follows. The monomer is modelled as a sphere of diameter d (volume V), with rotational relaxation time/90. The dimer is modelled as a prolate ellipsoid of rotation with minor axis d, major axis 2d (volume 2V). This ellipsoid of rotation has two rotational relaxation times, 191,corresponding to rotation about the minor axis, and 19~, corresponding to rotation about the major axis. Using the equations of Perrin [7,8], we calculate/91 = 3.0 19o and 192 = 2.1 190. The apparent rotational relaxation time of the ellipsoid of rotation, as determined from the fluorescence polarization via Eqn. 1, is an average of 191 and 192 which is weighted according to the orientation of the excitation and emission dipoles of the fluorophore(s) relative to the major and minor axes of the ellipsoid [12]. The apparent rotational relaxation time of an ANS-Mb dimer would thus be expected to lie between 2.1 and 3.0 times that of the monomer, in agreement with our interpretation of the data. In summary, the experimental results presented here are consistent with the hypothesis that ANS-Mb self-associates to a dimer in the presence of sufficient

322 concentrations of protein additives, and that the nature of this dimer is, to a first approximation, independent of the particular protein species employed as additive. We believe that this hypothesis provides the simplest interpretation consistent with both theory [1] and experiment. Acknowledgments The authors thank Dr. D. Atha of the American Red Cross, Bethesda, Maryland, and Dr. H. Edelhoch of the National Institutes of Health for gifts of materials, and Dr. Edelhoch for advice and criticism.

Appendix

~'M ~H ~'M(0) ~ rH "0~t)

Derivation of text Eqn. 3 Stryer [3] determined the value o f p ° to be equal to 0.396 for both ANS-Mb and ANS-Hb. This value is identical, to within experimental error, to the value of p measured in the present study at the freezing point, i.e., under conditions such that the rotational relaxation time is expected to be much greater than the fluorescence lifetime. Hence we shall set pM° = pH ° = p ° = 0.4. To a good approximation, this value may be assumed to be independent of the concentration of added macromolecular species [13,14]. Eqn. 1 may then be rewritten

1 251~ rM ~/~ ~M 1 _ 2.5 + \(p~v~(O)

,I~,rM(O) I/ \PM(O)/

PM

'

7H

PH

)

(A1)

PH where p (0), r (0) and p (0) represent the values of these parameters in the absence of added polymer or protein. The mean values of p~(0) and pu(0) obtained in the present work agree well with the corresponding values obtained by Stryer [3 ] under similar conditions. Given these values (see 'Results') and making the substitutions indicated in text Eqn. 2, Eqn. A1 may be rewritten

OM ~rM(0)]\ n ]1 \0M(0)!

1 _ 2.5 ÷ 3 . 2 1 1

PM

1 - 2.5 + 1 2 o I

OH

"

qT

OH

-))t-Tt

The fluorescence lifetime r is proportional to quantum yield, which is in turn proportional to fluorescence intensity [15]. We have observed that the fractional decrease in fluorescence intensity observed upon addition of lysosyme to ANS-Hb is the same, to within -+10%, as that observed upon addition o f an equal concentration of lysozyme to ANSMb. This result is not unexpected; the close similarity between the fluorescent properties of ANS bound to apoHb and apoMb [3] suggests that the quenching o f ANS-Hb and ANS-Mb fluorescence by a given macromolecular additive should be closely similar as well. We shall accordingly assume that for a given concentration of additive

(A2a) (A2b)

(A3)

Eqn. A2 and A3 may be combined to yield text Eqn. 3.

References 1 Minton, A.P. (1981) Biopolymers, in the press 2 Antonini, E. and Brunori, M. (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands, North-Holland, Amsterdam 3 Stryer, L. (1965) J. Mol. Biol. 13,482-495 4 Kinosita, K., Mitaku, S. and Ikegami, A. (1975) Biochim. Biophys. Acta 393, 10-14 5 Rossi-Fanelli, A., Antonini, E. and Caputo, A. (1958) Biochim. Biophys. Acta 30, 608-615 6 Chen, R.F., Edelhoch, H. and Steiner, R.F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S.J., ed.), Part A, pp. 171-244, Academic Press, New York 7 Pert'in, F. (1934) J. Phys. Radium Ser. VII 5,497-511 8 Koenig, S.H. (1975) Biopolymers 14, 2421-2423 9 Ross, P.D. and Minton, A.P. (1977) J. Mol. Biol. 112, 437-452 10 Ross, P.D., Briehl, R.W. and Minton, A.P. (1978) Biopolymers 17, 2 285-2 288 11 Ross, P.D. and Minton, A.P. (1977) Biochem. Biophys. Res. Commun. 76,971-976 12 Weber, G. and Anderson, S.R. (1969) Biochemistry 8, 361-371 13 Laurent, T.L. and Obrink, B. (1972) Eur. J. Biochem. 28, 94-101 14 Obrink, B. and Laurent, T.C. (1974) Eur. J. Biochem. 41, 83-90 15 Pesce, A.J., Rosen, C.-G. and Pasby, T.L. (1971) Fluorescence Spectroscopy, Marcel Dekker, New York