Properties of s100 protein studied by fluorescence methods

Biochimtca et Biophysi¢a A cta, 703 (1982) 231 - 240

231

Elsevicr Biomedical Press

BBA 31122

P R O P E R T I E S OF SI00 PROTEIN STUDIED BY FI,UORESCENCE M E T H O D S ROBERTO I). MORERO * and G R E G O R I O WEBER

Department ~t" BtochemistIT, School ~( ('hetm¢al Scram'e.g. Unit:er~ttv of Illmm.~, I 'rhuna, II. 61,~'01 /l'.S...t.i (Received September 29th, 198 l)

Ket' word~." S I (X) protetn; kTuorescem'e

The intrinsic fluorescence of the SI00 protein, due to both tyrosine and tO'ptophan, increases several-fold, in reversible fashion, in solutions at pH 3.0 in comparison with the neutral molecule. The study of the rotational diffusion of the photo-conjugates of I-azidonaphthalene-5-sulfonate with SI00 as a function of pH, the concentration-dependence of the fluorescence polarization and the electrophoretic patterns indicate that protein unfolding without dissociation into subunits takes place in the pH region 4-3.4 and that dissociation into subunits is complete in p. M solutions of the protein at pH 2.9. Anionic binding sites, probably connected with arginine residues, can be detected in acid solutions, as indicated by a 30-fold increase in fluorescence efficiency of added anUinonaphthalene sulfonate at pH 3.4, as compared to the efficiency at neutral pH. At pH 3.4 the protein can be transferred reversibly to an isobutanol or pentanol phase, not only by the addition of sodium p-toluenesulfonate, which is an effect observed for other globular proteins, hut by the addition of calcium chloride or magnesium chloride as well, a specific effect probably related to the function of this protein.

Introduction

Since the discovery of the SI00 protein, first isolated by Moore [1], different roles for the function of the protein in the nervous system have been proposed. SI00 is present in the brain of all the vertebrates and invertebrates investigated, and the serological activity is remarkably constant from species to species [2-5]. The molecular weights reported range from 21000 to 24000 [I-6]. Dannies and Levine [7] have proposed that the SI00 protein is made of three non-identical subunits of 7000 molecular ',,,'eight. It is interesting to remark * International Postdoctoral Fellow from Department of Health, Education and Welfare. Public Health Service. Present address: lnstituto de Quimica Biologica, Facultad de Bioqimica, Quimica y Farmacia, Universidad National de "l'ucum',in, San Miguel de Tucuman, Argentina. Abbreviation: ANS, I-anilino-8-naphthalene sulfonate. 0167-4838/82/0000-0000/$02.75 ..~ 1982 Elsevier Biomedical Press

that the protein is found in the soluble fraction of the brain, but a significant proportion is isolated with the microsomal fraction [8]. While the SI00 protein properties and their potential physiological significance are beginning to be documented, current knowledge is still fragmentary. Earlier studies by Moore [ 1], Calissano et al. [9] and Kessler et al. [4] have described the preparation and characterization of the SI00 protein. Many proteins undergo structural changes as the charge of the macromolecule changes with the pH of the solvent [10,11]. In many proteins decrease of pH leads to unfolding, more or less complete in the different cases. The results found in the literature indicate that the extent of the structural changes with pH is a characteristic property of the protein related to its structure and function. We report here studies of some of the physical and chemical properties of bovine brain

232 S100 protein subjected to ptl changes, and compare its behavior to that of other proteins. ()ur objective was to obtain further information on the protein to provide a basis for investigation of its physiological role. M a t e r i a l s and M e t h o d s

Protein purification. Bovine S100 protein was purified essentially by the m e t h ~ of Dannies and Levine [7] with minor modifications. Briefly. the purification procedure consists of the homogenization of the bovine brain suspended in 5 mM TrisP()~ buffer (pH 7.3)./5 mM EDTA. The homogeniled material was centrifuged twice at 20000 × g for 60 rain. "[he supernatant was subject to ammonium sulfate fractionation. Solid salt was added to it to make a 90% saturated solution. After stirring for 60 min the preparation was centrifuged as described above. The ammonium sulfate concentration was then adjusted to 98% saturation and the pH brought to 4.0 with concentrated hydrochloric acid. The solution was stirred for 2 h and the precipitate collected by centrifugation, was resuspended in 5 mM Tris-PO~ buffer, pH 7.3, 5 mM EDTA and dialyzed overnight against the same buffer. The dialyzed material was applied to a DEAE-ccllulose column equilibrated with the above-mentioned buffer and eluted according to the method of Uyemura et al. [12]. The fractions containing the SI00 protein were pooled, concentrated by molecular filtration through a pellicon membrane (Millipore), and passed through a Sephadex G-100 column equilibrated and eluted with 50 mM sodium phosphate buffer (pH 7.3)/5 mM F,DTA. The fraction containing the S100 protein were dialized 24h against distilled water and lyophilized. The powder was stored at -- I5°C until used. The final preparation examined by 10% polyacrylamide gel electrophoresis showed only one band as detected by Coomassie brillant blue 250 staining of the heavily overloaded gels. Preparation of solutions. Solutions of purified bovine SI00 protein were prepared by dissolving lyophilized material in 20 mM Tris-HC1 buffer. p i t 7.2. The solutions were dialyzed 10 h against the same buffer and centrifuged at 5000 rpm for 5 rain in order to clarify them. Lower pH values were obtained by the careful addition of small

amounts of 2 M HCI from a Hamilton syringe while the solution was stirred. Protein concentrations were determined from absorbance measurements at 280 nm assuming a molar extinction coefficient of 8260 tool ~. cm J [9]. or by' the method of Lowry et al. [13]. Label#n+ procedure. Bovine SI00 protein was labelled by photoreaction with the sodium salt of l-azidonaphthalene-5-sulfonate in 20 mM TrisHCI buffer, pH 7.3. The mixture, containing 0.05 mM protein and 0.1 mM azido compound, was irradiated for 10 rain at room temperature with an ultraviolet lamp emitting predominantly at 366 nm, and finally passed through a small column of Sephadex G-25 in order to remove all the free dye from the conjugate. The molar ratio of azido compound to protein ranged from 0.5 to 1.0. l-Azidonaphthalene-5-sulfonate was prepared from the corresponding amine by a method developed in our laboratory (unpublished data). Physical measurements. Absorption spectra and difference absorption spectra were recorded in a Beckman MVI spectrofluorometer. Fluorescence emission spectra were recorded with an emission spectrometer equipped with bipolar averaging circuit and digital integrator [14,15]. Fluorescence life-times were measured on the phase-modulation cross-correlation fluorometer described by Spencer and Weber [17]. Measurements of fluorescence polarization were made on a polarization photometer previously described [18]. The dependence of the fluorescence polarization ( P ) upon the rotation relaxation time (p) and the lifetime of the excited state ( r ) is given bv the Perrin equation, where t]~ is the limiting polarization (l , ' t ' - - I / 3 ) - ( I / P .

1,;3)(I ~- 3~'/p)

Since p is directly proportional to n,'l" (the ratio of the viscosity to the temperature of the solution in K), a plot of I / P against T/n gives a straight line with intercept 1/'P0. and slope from which the rotational relaxation times can be calculated. Results

Effect of ptl on the tntrm~tc fluorescence of SIO0 protein The molecule of SI00 proteins has only one

233

tryptophan residue and three tyrosine residues [5]. When the native SI00 is excited by 275 nm radiation, the fluorescence contains emission by both tyrosine and tryptophan residues. On excitation by light of 295 nm, the emission is only due to tryptophan, as tyrosine does not absorb appreciably this wavelength. Therefore, by exciting with 295 nm radiation, a pure tryptophan emission spectrum is obtained, while the sum of tyros~ne and tryptophan emission is obtained by exciting at 275 nm. Analysis of the components of the fluorescence emission into tyrosine and tryptophan contributions is made possible by normalizing the pure tryptophan spectrum at 370 nm where there is no appreciable contribution from tyrosine, and obtaining the difference of the fluorescence spectra obtained by excitation at 275 and 295 nm [19]. Fig. 1 shows a typical intrinsic fluorescence spectrum of SI00 at pH 3.5, and the tryptophan and tyrosine contributions. At this pH, 60% of the total fluorescence corresponds to tryptophan and 40% to tyrosine. The tryptophan and tyrosine fluorescence and the sum of both (total fluorescence) was measured when a neutralized solution (pH 7.3) was either acidified to pH 3.0 or brought to

pH I1.0. A plot of the integrated fluorescence intensities as a function of pH is shown in Fig. 2. Changes were minor between pH 5.0 and 8.0. At pH 7.2 the major fluorescence component corresponds to tryptophan emission (about 80%). As the pH diminished from 4.0 there was a sharp increase in both the tyrosine and tryptophan yield. As the pH was raised from 8.0 tryptophan emission increased gradually, while the tyrosine component became almost zero. The latter is to be expected because of the negligible fluorescence yield of tyrosinate ion. Back titration from pH 3.0 or pH 11.0 to neutral pH showed complete agreement with the initial values, indicating the reversibility of the observed effects. Only a slight change in the position of the maximum of the fluorescence of tryptophan was, however, detectable at differ-

.04 w u

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350 460 WAVELENGTH (nm) Fig. I. Intrinsic fluorescence emission spectra of SI(R) protein at pH 3.5 {solid line). Protein concentration was 0.6 mg/ml, . . . . . , tyrosine contributions: . . . . . . , tryptophan contribulion. Buffer. 20 mM Tris-HCl.

.0~

2~o

g pH

z&o

3bo

WAVELENGTH(rim)

8

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Fig. 2. Relative fluorescence emission of SI(R) protein as a function of pH. • .. • , tyrosine fluorescence, {2 2. tryptophan fluorescence: O O. total fluorescence. Inset: the ultraviolet difference spectrum of SICK) protein at pH 3.0 (reference was at 7.2) in 120 mM Tris-HC1 buffer. Protein concentration. I mg/ml. Temperature, 25°C.

234 ent p H values (not shown). A shift of 5 - 7 nm to the blue is observed in the acid solutions and a shift to the red ( 5 - 8 nm) of similar m a g n i t u d e in the alkaline solutions. The fluorescence polarization of the ultraviolet emission ( 3 0 0 - 4 0 0 nm) of the S100 protein on excitation from 250 to 300 nm was studied at p H 7.3, 3.4 and 10.5. The polarization values at p H 3.4 and 7.2 differ only slightly, but the lifetime at acid p H was almost twice the value at neutral p H ( T a b l e l). A t p H 10.5 a large decrease in the polarization value was measured. F r o m the polarization and fluorescence lifetime at 25°C we calculated the a p p a r e n t rotational relaxation times of the system to be 14.8 ns and 3.6 ns for SI00 at p H 3.4 and 7.2, respectively. The rotational relaxation times derived from the polarization of the t r y p t o p h a n fluorescence are an o r d e r of m a g n i t u d e smaller than the values that c o r r e s p o n d to a protein of the mass of SI00, and reflect mainly a local rotational motion of the t r y p t o p h a n [20]. This motion if completely, free would yield unpolarized fluorescence, and the existence of a p p r e c i a b l e polarization has therefore to be assigned to the restricted m o t i o n of the trypt o p h a n which is confined, at a fixed temperature, to an average a m p l i t u d e of rotation ( 0 ) given by the equation:

(1/t'--1/3) (I/P.

- I/3)

=lq-

3r

2

oh

3 cos20- 1

or

cos20-1/3

I

2

I±

I+

a, .,, rob

Using the d a t a of T a b l e I this equation gives ( 0 ) -~ 36 ° at p H 7.2 and ( 0 ) > 30 ° at p H 3.4. Although a fraction of these rotational a m p l i t u d e s may result from overall m o t i o n of the protein, the bulk of it (25 30°), is to be a t t r i b u t e d to local motions. which are less affected than the overall protein rotations (see below) by the p H changes.

Ultrae~iolet absorption spectra. A significant change in the ultraviolet a b s o r p t i o n of the SI00 protein occurs when the p H of the

"I'AtYI,E1

ROTATIONAl. RELAXATION TIMES OF SIO0 FROM POI.ARIZATION OF THE INTRINSIC FLUORES('ENCt.~ pI1

211:( 1 >, b....

I'd'

r {n~,)~'

p (n>)'

3.4 7.2 10.5

0.175 0.140 0.090

0.2g 0.2g 0.2g

3.2 1.3 1.9

14.S 3.6 2.5

" l.imiting polarization taken as the polarization observed for solutions of SI00 in 80% propy,lene glycol at -- 15°(" S.I). of polarization:,: • 0.(X)2. i, Phase liDtime~,.S.D.: " 0.05 m,. ' Rotational relaxation time calculated from Perrin equation. S.I). ' 0.3 ns. solution is brought from 7.3 to 3.5 (not shown). T h e differential ultraviolet spectrum between solutions at p H 7.3 and 3.5 gave two m a x i m a at 280 and 287 nm, respectively (inset. Fig. 2). Most of the change in the ultraviolet a b s o r p t i o n was completed in the time required for the measurements. These results are interpreted as indicating a hydrogen b o n d between a tyrosine residue and a carboxyl ion acceptor group which is broken by' protonation of the carboxvlic ion in acid. The same characteristic differential spectrum was found with ribonuclease at p H 6.94 and 1,91 [21], insulin at p H 7.0 and 2.0 [22], and bovine p l a s m a a l b u m i n at neutral and acid p H [23]. To d e t e r m i n e whether the spectroscopic changes measured between p H 5.0 and 3.0 reflected other changes in the size or shape of the SI00 molecule, we studied a h y d r o d y n a m i c p a r a m e t e r : the rotational relaxation time of conjugates of 1a z i d o n a p h t h a l e n e - 5 - s u l f o n a t e with S100, anti the electrophoretic b e h a v i o r of the native S100 at different pH values.

l"luor¢~cence of the bot'ine SIO0 conjugates. The variation of the fluorescence polarization with p H of a z i d o n a p h t h a l e n e conjugates of the SI00 p r o t e i n is shown in Fig. 3 (inset). A marked increase takes place when the p H of the solution decreases from 5.0 to 3.5, the transition zone observed by' the previously discussed procedures. In a d d i t i o n to this effect, a large decrease in the p o l a r i z a t i o n occurs at p H lower than 3.2 and higher than 8.0.

235

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from 3.5 to 3.2 where evident curvature is present. The fluorescence lifetime of the azido conjugate of the SI00 protein at different pH and temperaturcs is shown in Table II. N o modifications in the lifetime values werc found between 0 to 25°C from pH 7.3 to 3.4 in all cases. At pH lower than 3.0 the lifetime incrcased slightly. Fig. 4 shows a plot of the rotational relaxation time at 25°C, calculated from the Perrin equation, against the pH of the solution. The values obtained at pH 3.4 are almost 4-fold greater than those obtained at pH 7.3. At pH lower than 3.0 the rotational relaxation times dropped abruptly. The probable dissociation of the protein into subunits at pH lower than 3.0 was evaluated by following the effect of dilution upon the polarization of fluorescence of the azidonaphthalene sulfonate conjugate of SI00 at pH 3.8, 3.4 and 2.9. As shown in Fig. 5, no measurable changes in the polarization of fluorescence of the complex were found at pH 3.8 and 2.9. The titration curvc

T/'I'] x I0 - 4 as a function of 7"/, for the conjugate SI(X) protein and I-azidonaphthalene-5sulfonate, in 20 m M Tris-IICI buffer, p l l 7.3 ( 0 O). pH Fig. 3. Reciprocal of polarization ( l / P )

6.9 ( O O), p l t 6.0 (X XI, pH 3.5 ( I I), pH 2.9 l.& /',). Inset: the influence of pH on the polarization c,f fluorescence of I-5-azido compound-conjugate SI00. Protein concentration, 0.02 m g / m l . Buffer. 20 m M Tris-HCl. The p t t was adjusted with ItCI. Temperature, 25°C. Excitation wavelength, 340 nm. The fluorescence was passed through a 2 M N a N O , filter.

The polarization of the azidonaphthalenc conjugate of SI00, as a function of the quantity T/n (temperature K/viscosity centipoise) at different pH, is givcn in Fig. 3. The viscosity of the solutions was changed by decreasing the temperature from 25 to 0°C. To covcr the range of T/n values smaller than that accessible in water solutions, the viscosity was adjusted by varying the anaount of sucrose added to the solution. Measurements were carried out aftcr the equilibrium was rcached. Polarization values stabilized after a few minutes at pH values greater than 3.4, but it was necessary to wait almost 2h when the pH was lower than 3.2. A good linearity of the Perrin plots was obtained at all pH values with exception of the range

T A B L E I1 ROTATIONAL NAPHTHALENE

RELAXATION TIMES OF AZIDOS U L F O N A T E C O N J U G A T E O F SI00

Lifetimes (r) were obtained by modulation at 30 mHz. Limiting polarizations were calculated by extrapolating Perrin plots. The rotational relaxation times, p2~ are those measured in water solution at 25°C or reduced to this temperature and viscosity if made in another solvent (d). Na-Pts, s o d i u m ptoluene sulfonate. Water solution

"1"

P.

pH 7.2 pll 3.5 initial slope final ,,,lope pIl 2 9 no adddition plus 5 m M

19.02 ~ 0.10

(I.29

43.O

I g.93 -" 0.15 18.93 • 0.15

0.29 0.27

460 184.2

20. I I -' O. I I

O. 19

52.3

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20.6X • O. I I

0. I q

125.0

21.12 • 0.15

0.19

102.0

14.08 • 0.17

0.21

Na-Pts

Oh

plus 50 mM (.'aCI, plus 200 m M MgCI 2

Isobutanol (water-,,,aturated :it 0"C) plus 5 mM Na-Pts

16.5 (d)

236

changes correspond 4 . 5 . 1 0 *M. S

l

w I

Z

i-el x <

~ 2~. .J Z 0 I- I00' I--. o elL,"

3 i

i

pH

Fig. 4. pH profiles or rotational relaxation time of bo,.ine SI(X) protein conjugate to I-azidonaphthalene-5-sulflmate in 20 mM Tris-H('t buffer. The rotational relaxation time ",,.as calculated from the Perrin equation (see Materials and Methods). ptl effect on the SI(X) partitions between water phase and isobut~,lalcohol. Equal volumes of v, ater phase (20 mM Tris-tlCI. 5 mM p-toluene sulfonate) and organic phase '.',ere mixed genii) (3 5 rain) and after both phases became ,,,eparate and clear, fluorescence emission v, as measured in the organic phase. Excitation v, avelength, 340 nm.

obtained at pH 3.4 shows maximum polarization values at protein concentration higher than 10 6 M, and a gradual drop at concentration lower than 1 0 7 M. The mid-point of the polarization

to a

protein concentration near

Ele<'trophore.~'is o/SIO0 protein at d(l'fi'rent pll calIlC~, "

Fig. 6 shows how the bovine S100 protein migrated in an electric field at three different pHs. At pH 7.2 only one anionic band could be detected on the cellulose acetate slide, while at pH 3.0 three bands appeared, migrating in the opposite direction. Above pH 7.2 only one band was also seen. This became broader as the pH w.as increased. The results of the electrophoresis can be correlated with the observations of fluorescence polarization. At pl-! 7.2 3.4 the protein exists (in the concentratkm range used in the experiment: 10 p.M 1 nM) as the native aggregate. At pit 2.'-) it exists in the dissaggregated form over the same range of concentration. At p|! 3.4 it is full\' aggregated at concentrations greater than 10 ~.M but it disaggregates readily on dilution, reaching the same state that is found at pH _,.9. when the concentration is 1 nM. From the midpoint of the effect one can calculate a free energy change of approximately 10 Kcal-mol i upon formation of tile native aggregation from the subunits at pH 3.4. The absence of appreciable ct, rvaturc in the

(+)

(-)

P

pH 7.2

0.2C

pH 1~.9 0.15

' O.IC

pH 10.5

'1'

ORIGIN

4,

--}

4

-~,

LOG O=ROVtI~J Fig 5. Plot of polari;,ation value of I-azidonaphthalene-5sulfonate conjugate to SI(R) protein as a function of complex concentration.

Fig. 6. Eleetrophorcsis of bovine SI(X) protein on cellulose acetate ( M i l l i p o r e Corp, Bedford. M A . or [ telena Laboratories,.

]~¢aumont, TX) performed at 25~(." in 5 mM l'r>-H('l buffer. plt 7.2 (top), pH 3.0 (middle) and pll I0.0 (bottom) The •,ohage applied wa~, 60 V. The slides '.,,'ere stained for prol,:ln with ICi ('oomassic brillant blue in 7r:,: acetic acid and deMained b?~ v~a,,,hing 'aith 7q; acetic acid.

237

Perrin plot at pH 7.2 indicates that the protein has a 'globular shape' at neutral pH. The disaggregated form (pH 2.9) is also, within the gross limits given by fluorescence polarization, a globular molecule. On the other hand. the high polarization form seen at pH 3.5 shows very evident curvature of the Perrin plot. This indicates the presence of two or more appreciably different rotational rates, arising perhaps from the combination of the overall motion of the protein and increased local freedom of the fluorophorc.

't 2

Binding of l-anilmonaphthalene-8-sulfonate to SIO0 ANS becomes highly fluorescent upon binding to the hydrophobic sites of proteins. The structural changes found in the SI00 protein at low pH may bc expected to result in the appearance of hydrophobic regions hidden in the native, neutral pH, structure. The ability of SI00 to bind ANS was determined in solutions of various pH values. Although a very weak fluorescence of ANS was detected in the presence of S100 protein at neutral pH (the same result was obtained by Calissano ct al. [9]), acidification of the solution enhanced the ANS fluorescence almost 35-fold. The pH dependence of the ANS fluorescence shown in Fig. 7 revealed a sharp transition of the SI00 protein occurring between pH 4.0 to 3.0, and a second one at pH lower than 3.0. These correspond quite well to the change in shape and the dissociation revealed by fluorescence polarization and elcctrophoresis. As shown in Fig. 7, the binding of ANS to SI00 was significantly decreased after treating the protein with glyoxal or acetic anhydride. This results suggests that arginine residues at the SI00 protein provide the electrostatic binding at low pH, a situation that may be common to most cases of strong anion binding by proteins [24,25]. Partition of the SIO0 protein between water and isobut.vlalcohol phases The binding of ANS to native SI00 protein indicated that protein hydrophobic sites became exposed as the pH was decreased, reaching a maximum at pH around 3.2. To confirm the abovementioned results, partition studies of the protein between water and isobutylalcohol, a non-polar solvent, were carried out [26]. When S100 in 20 mM Tris-HCl brought to pH 3.5 is partitioned

pH Fig. 7. Effect of pH and SIO0 protein on ANS fluorescence. Relative fluorescence of a solution of 20 mM Tris-HCI buffer, A N S (10/*M), and SI00 (10 p,M). Excitation wavelength, 385 nm. Temperature. 25°C. • • , SI(X) without treatment. • II, SI00 treated with acetic anhydride: • •. SI(R) treated with glyoxal.

with an equal volume of isobutylalcohol all the protein remains in the water phase. Strikingly, in the presence of 50 mM CaCI 2 all the SI00 protein is transferred to the upper phase (isolbutylalcohoi). The effect of other ions such as Mg 2~ , N a t , K ~ and sodium p-toluene sulfonate on the partition of the protein is shown in Fig. 8. The anionic ptoluene sulfonate (sodium salt) was the most effective ion. Mg 2~ was also able to promote the transfer of the protein to the organic phase, but concentrations as high as 200 mM were required. Both monovalent cations, N a " and K ' , had the same effect on the partition of the protein between the two phases, but is is interesting to note that with them a different behavior was seen when Ca 2 ~ or Mg 2~ were present: only 30% of the protein could be transferred to the isobutylalcohol phase, the rest remaining at the interphase. The effect of the pH on the partition at constant concentration of p-toluene sulfonate is shown in Fig. 4. The protein began partitioning at pH 4.0 and around pH 3.2 all the protein was found in the upper phase. Identical results were obtained using n-pentanol instead of isobutylalcohol. All the partition experiments were carried out at room

238

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_

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ION CONCENTRATION (raM)

t:ig. 8. Effect of Ca;" (Q Q), Mg ~'' (/J -[5), ptoluene sulfonate (C,) O), K ' and Na" (,& A) concentrations on the SI00 partition between water phase (pH 3.5) and isolbutylalcohol. The S100 conjugate to the lazidonaphthalene-5-sulfonate in 20 mM Tris-HCl buffer, pH 7.2, was brought to pH 3.5 and in the presence of the mentioned ions mixed gently (3-5 rain) with an equal volume of isobutylalcohol. After both phases became dear, fluorescence emission was measured in the upper phase. Excitation wavelength. 340 nm.

temperature (23-25°C), and fully reversible behaviour was observed upon changing the pH. Perrin plots of SI00 protein in aqueous acid solution (pH 2.9) without additions and in the presence of CaCI 2, sodium p-toluene sulfonate or MgCI 2 are shown in Fig. 9. The values of the rotational relaxation times increased from 45 to 125 ns with C a - " , or to 105 ns with p-toluene sulfonate. A non-linear plot was observed at pH 2.9 in the presence of 200 mM MgC12. The Perrin plots of SI00 conjugate, in the presence of ptoluene sulfonate, in the upper phase, showed a rotational relaxation time, reduced to the viscosity of water, of only 16 ns, indicating the appearance of additional rotational modes in the protein in the upper phase. As shown in Fig. 4, the pH profile of the partition of the Na + p-toluene sulfonate-protein complex resembles the observed pH profiles of fluorescence emission, rotational relaxation time and ANS binding, indicating that all these effects are dependent upon changes in the structure a n d / o r charge that take place upon reducing the pH.

41 ,io

J.o

3'.o

T/7'} x 10 - 4

Fig. 9. Reciprocal of polarization ( I / P ) as a function c,f l,, n for the I-~.idonaphthalene-5-sulfonate COnlugate to, SllX) protein, in 20 m M Tris-HCI buffer, ptl 2.9, with no addition 1/', /X), plus 200 mM Mg-" ( ' J !]1. plus 100 mM ( ' a 2 " ( 0 0 ) , plus 5 mM sodiump-toluene ~ulhmate (0 O), and in isobutanol (X X).

Discussion and Conclusion

The various methods employed indicate clearly that, as the pH decreases, two changes taken place in S100 protein. The first is characterized bv a large increase in the fluorescence efficiency of tryptophan and tyrosine, changes in differential absorption indicating breakage of one or more tyrosine-carboxylate bonds, large increases in rotational relaxation time of the azidonaphthalene conjugate of the protein, the appearance of strong binding sites for anilinonaphthalene sulfonate and the ability of the protein to transfer to a relatively non-polar medium in the presence of salts. The reversible aggregation of the system, at pH 3.4, is demonstrated by the concentration-dependence of the fluorescence polarization. At still lower pH value a sharp decrease in rotational relaxation time. insensitivity of the fluorescence polarization to dilution, and electrophoretic heterogeneity dem-

239

onstrate that the actual dissociation into subunits is complete. The increase in fluorescence efficiency of tyrosine and tryptophan must be attributed to the removal of quenching groups from the neighborhood of these residues. The lengthened rotational relaxation time requires a structure in which partial rotation motions have an amplitude which is not materially greater than in the neutral species. This feature is particularly well-substantiated by the finding that tryptophan exhibits rotational motions of similar amplitude at pH 7.2 and 3,5. A model able to represent the observed feature is shown in the scheme of Fig. 10. The additional areas of contact in Form A would provide for the reduced fluorescence efficiency at neutral pH. The importance of electrostatic repulsion in the generation of the expanded forms B and C is evidenced by their appearance at both high and low pHvalues. The transfer of the protein at low pH to buffer-saturated isobutanol or n-pentanol under the influence of the hydrophobic anion sodium p-toluene sulfonate is to be expected, as it is observed in other globular proteins [26]. The transfer under the influence of CaCI 2 and MgCI 2 alone represents clearly a more specific effect which requires further study for its characterization. The increased rotational relaxation time observed upon addition of Ca 2 ~ or Mg 2 ÷ , or p-toluene sulfonate. indicates that the species transferred to the organic phase is an aggregated form, perhaps the neutral oligomer. In any case, the charge of the protein complexes in the upper phase must be fully neu-

A

B

o~ 7

~H 3.4

A~55n~

2~80n~

C

D. a8

p ~ 6 0 o~

Fig. 10. Schematic drawing showing the molecular change of SI(X) protein at different pH values. A represents the native molecule with its three different polypeptide chains. B, denaturalized structure with the polypeptide chains ~till bound together. C, the disscociated polypcptide chain.

tralized, so that we are led to the conclusion that binding of the divalent cations must result in binding of anions beyond what is required for their own neutralization, thus defining an interesting structural problem. This is characterized by exposure of hydrophobic groups, and in the light of published observations of the association of SI00 with liposomes [27] it is reasonable to suppose that it may be important to the functions of the protein.

Acknowledgements This investigation was supported in part by a Public Health Service International Research Fellowship (I-F05-TW-2224-01)and by a Grant G M 11223 from The National Institute of Health.

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