Structural requirements for steroid binding and quenching of albumin fluorescence in bovine plasma albumin

Biochimica et Biophysica Acta, 439 (1976) 175-193

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37370 S T R U C T U R A L R E Q U I R E M E N T S FOR STEROID B I N D I N G A N D QUENCHING OF ALBUMIN F L U O R E S C E N C E IN BOVINE PLASMA A L B U M I N ~

A. MARGINEDA ROMEU, ELBA E. MARTINO and A. O. M. STOPPANI CStedra de Quimica Biol6gica, Facultad de Medicina, Paraguay 2155, Buenos Aires (Argentina)

(Received October 14th, 1975) (Revised manuscript received February 27th, 1976)

SUMMARY 1. Steroids interact with bovine plasma albumin at a binding region that involves tryptophanyl, tyrosyl, arginyl and lysyl residues. The function of the tryptophanyl residues is demonstrated by: (a) the decrease of albumin binding affinity after modification of one tryptophanyl with 2-nitrophenylsulfenyl chloride; (b) steroid quenching of albumin tryptophanyl fluorescence; and (c) steroid quenching of 1anilinonaphthalene-8-sulfonate fluorescence, when it is excited by energy transfer from excited tryptophanyls. The function of tyrosyl residues is demonstrated by the decrease of albumin binding affinity after nitration of 30 ~o tyrosyls with tetranitromethane, or deprotonation of tyrosyls by variation of pH. The function of arginyl and lysyl residues is demonstrated by the decrease of binding affinity after modification of these residues with glyoxal, formaldehyde or acetic anhydride. The presence of both apolar (Trp, Tyr and Lys (deprotonated)) and polar (Arg and Lys (protonated)) residues at the steroid binding site fits in well with the site relative apolarity, when expressed on the Kosower scale (Kosower, E. M. (1958) J. Am. Chem. Soc. 80, 32533260). 2. The contribution of specific amino acid residues to steroid binding depends to some extent on the steroid structure, as exemplified by the quantitatively different role of arginyl (or lysyl) residues in albumin interaction with testosterone acetate and epitestosterone, respectively, or that of tyrosyl residues in albumin interaction with 11-deoxycorticosterone and epitestosterone, respectively. 3. The concerted action of polar and apolar amino acid residues is an essential requirement for steroid binding, since unfolding of albumin polypeptide chain by guanidine-HC1, urea, or by reduction of disulfide bridges with 2-mercaptoethanol,

Abbreviations and steroid nomenclature: l l-deoxycorticosterone, 21-hydroxy-4-pregnene3,20-dione; epitestosterone, 17a-hydroxy-4-androsten-3-one; testosterone acetate, 17fl-hydroxy-4androsten-3-one 17-acetate; androsterone, 3a-hydroxy-5a-androstan-17-one; cortisol, 11fl,17a,21-trihydroxy-4-pregnene-3,20-dione;prednisone, 17a,21-dihydroxy-l,4-pregnandiene-3,11,20-trione; corticosterone, 11fl,21-dihydroxy-4-pregnene-3,20-dione;other steroid names are in accordance with the systematic nomenclature. ANS, 1-anitinonaphthalene 8-sulfonate. "This work was taken in part from a Thesis submitted by A. Margineda Romeu to the Universidad Autonoma de Barcelona (Spain), for the Degree of Doctor of Chemistry (1973).

176 strongly decreases steroid binding to albumin while, conversely, reoxidation and refolding of the unfolded polypeptide chain restore albumin affinity for steroids. 4. Parallel determinations of steroid binding constants by equilibrium dialysis and fluorimetric titration, as well as the general pattern of the pH and temperature effects on steroid quenching of albumin fluorescence, confirm the validity of the fluorescence quenching titration as an effective method for measuring albuminsteroid molecular interactions.

INTRODUCTION Hormonal steroids and related compounds form dissociable complexes with plasma albumin that play an important function for the transport of steroid hormones by the blood [1 ]. It is postulated that these complexes involve ligand-stabilized conformations [2], as suggested by polarimetric observations [3] and the modification of the rate of tryptic hydrolysis of albumin [4]. In contrast to the extensive information available on the molecular properties of steroids that determine their binding to albumin [5, 6], little is known about the region in albumin that interacts with steroids (the "steroid binding site"). Studies with bovine and human plasma albumins suggest that the site is characterized by the predominance of accessible hydrophobic amino acid residues, in particular tryptophanyl and tyrosyl residues, since: (a) steroids perturb the indole [7] and phenol [7] chromophores of albumin; (b) steroids form complexes with tryptophan and tyrosine ethyl esters [8] ; (c) steroids quench tryptophan and tyrosine fluorescence, and albumin tryptophanyl fluorescence [9]; and (d) ketonization of tyrosyl residues decreases albumin affinity for steroids [10]. In addition to its intrinsic physicochemical interest, full knowledge of the steroid-albumin interaction may prove relevant to ultimate understanding of steroid interaction with enzymes [11-13] and with specific binding proteins, particularly with "receptors" [14]. These circumstances prompted us to reexamine the mechanism of steroid binding to albumin, and particularly the role of specific amino acid residues and albumin folding state. Steroid binding to albumin was measured, when possible, by fluorescence quenching titration, a method recommended by Attallah and Lata [9] as very convenient for the study of steroid-protein molecular interactions. Albumin intrinsic fluorescence primarily depends on tryptophan [15] and, therefore, fluorescence titration is a suitable procedure to detect interactions at or near albumin tryptophanyl residues. MATERIALS AND METHODS

Reagents Crystallized bovine serum albumin, essentially fatty acid free (henceforth albumin), steroids and Tris were obtained from previously indicated sources [6]. The initial concentration of albumin solutions was determined by absorbance at 279 nm, assuming a molecular weight of 67 000 and an A~ cm value of 0.67 [16]. Steroids were added dissolved in redistilled ethanol, or in 0.1 M Tris.HCl buffer, pH 8.0. The latter solutions, that were used with the system albumin-ANS, were prepared as described by Ryan [17] and the steroid concentration was determined spectrophotom-

177 etrically, as described by Westphal [18]. Other reagents were obtained from the sources indicated: urea, guanidine.HC1 (enzymatic grade), from Mann Research Laboratories, Inc., New York; 8-hydroxyquinoline, 5,5'-dithiobis(2-nitrobenzoic acid), 2-mercaptoethanol and N-bromosuccinimide, from Sigma Chemical Co., St. Louis, Missouri (N-bromosuccinimide was recrystallized before use); 2-nitrophenylsulfenyl chloride and tetranitromethane, from Fluka A.G., Buchs; glyoxal and formaldehyde, from J. T. Baker, Phillipsburg, New Jersey; acetic anhydride from E. Merck, Darmstadt; trinitrobenzenesulfonic acid and D-10-camphorsulphonic acid from Eastman Organic Chemicals, Rochester, New York; 1-anilinonaphthalene 8sulfonate, from Serva Entwicklungs Labor, Heidelberg; Sephadex G-25, G-150 and DEAE-Sephadex A-50, from Pharmacia, Uppsala; ethanol was redistilled for 60 min under reflux in the presence of zinc powder (20 g/l) and potassium hydroxyde (40 g/l) before use. Glass-distilled, deionized water was used throughout. Fluorimetric measurements

Attallah and Lata [9] procedure was followed, as described before [6]. Unless otherwise stated, the fluorescence of albumin solutions was measured at 285 nm (excitation) and 342 nm (emission) in an Aminco Bowman spectrofluorimeter equipped with a cell compartment thermostated at 20 °C. 1.2 ml of 12-15 # M albumin solutions in 0.10 M Tris. HC1 buffer, pH 8.0, was placed in l-cm light path silica cuvettes. The fluorescence after each steroid addition (1-5/A) was measured and corrected for dilution from a blank titration with ethanol. The binding parameter Kf (formation constant of the steroid albumin complex) is defined by Eqn. 1 *

Of

Kf =

(1 -- Qr) ([St] - nQf. C)

(1)

where Qf is the quenching fraction (ratio of the quenching at, or near, the stoichiometric point to the maximum quenching); n is the molar ratio of steroid (free plus bound) to albumin at the stoichiometric point; C is the total molarity of protein at a certain point on the titration curve (corrected for dilution by the titrant); and [St] is the total concentration of steroid added. A typical titration curve can be seen in the preceding paper [6]. The Kr values presented are the mean of not less than 3 measurements. The free energy of formation (AG) of steroid-albumin complexes was calculated from the corresponding Kf values. An IBM 360 computer was used for the calculations. The actual deviation of the fluorimetric method is documented by the following Kf q-- average deviation values ( × 10 -4 (M-l)): androsterone, 12.1 4- 1.3; corticosterone, 15.1 ± 0.8; cortisol, 6.5 4- 0.2; epitestosterone, 8.0 4- 0.2; 17e-hydroxy-lldeoxycorticosterone 21-acetate, 14.3 4- 1.3; 17fl-hydroxy-17tt-methyl-4,9(11)-andro-

* In Attallah and Lata's original paper [9], Eqn. 1 appears printed as follows Kr = (1

--

Qt Qt) ([St] - nQt) C

which includes an error factor. In the preceding paper by Margineda et al. [6] the corrected equation (Eqn. 1 above) was used.

178 stadien-3-one, 11.1 ± 0.4; testosterone, 10.8 ± 0.6, and testosterone acetate, 27.7 ~_ 1.7. The maximum relative deviation was about 10~o, as exemplified with androsterone and 17a-hydroxy-ll-deoxycorticosterone 21-acetate. However, with 5aandrostane (a steroid omitted from the present study) the maximum relative deviation was about 25 ~o- The deviations presented above are consistent with those reported by Attallah and Lata [9] for similar titrations of steroid-albumin interactions. Apparently, deviation was related to steroid structure, and with the less soluble steroids an increase of turbidity of the albumin solution became an additional source of error [9]. Similarly, deviation occasionally increased when the same steroid was assayed with different albumin samples (see for example the effect of 17a-hydroxy-11-deoxycorticosterone 21-acetate in Tables II and IV), thus proving the need for adequate control measurements in every case. Fluorescence spectra were measured with the Aminco Bowman spectrofluorimeter attached to an x-y Moseley Autograph. Measurement of fluorescence polarization was performed as described by Velick [19].

Equilibrium dialysis measurements These were performed as described by Westphal et al. [20]. Dialysis bags were filled with 10 ml of 2 0 # M albumin solution in 0.10 M Tris.HC1 buffer, pH 8.0. The bags were immersed in 20 ml of 0.10 M Tris.HC1 buffer placed in stoppered glass tubes. Equilibrium was attained by shaking the tubes for 24 h at 25 °C. Steroid concentration in the outside fluid was determined by measuring the absorbance at 248 nm with the control (steroid-free) samples as blank. The molar extinction coefficient of each steroid under the given experimental conditions was determined as described by Westphal [18]. The binding parameters were calculated from Eqn. 2 1

1 --

n. K[S]

1 -t- -

(2)

n

where v is the mean number of steroid molecules bound per molecule of albumin; n is the number of binding sites; K the formation constant of the steroid-albumin, and IS], the molar concentration of steroid (free) inside the bag [21]. The plotting of 1/~ against 1/[S] gives a straight line; the intercept on the ordinate axis yields l/n and the slope of the line is 1/n.K.

Circular diehroism Spectra were taken on a Jasco model J-20 spectropolarimeter, precalibrated with D-10-camphorsulphonic acid. Measurements were performed at 20 °C, in a 1.00 mm path-length silica cell; albumin concentration was 1.0 mg/ml in 0.1 M Tris. HCI, pH 8.0. Spectra were recorded from 260 to near 200 nm. The aproximate helical content of the samples was estimated from the absolute values of the ellipticity at 221 nm. The ellipticity values for 100~ a-helix and 100~ random coil obtained for poly(L-glutamic acid) at 222 nm were used as limiting values in Eqn. 3 a-helix from [01221 =

[ [01221 [ Jr- 4800

45 400

× 100

(3)

where [01221 is the absolute value of the ellipticity at 221 nm. Other conditions were as described by Bewley et al. [22].

179

Preparation of albumin derivatives (a) Nitration with tetranitromethane was performed essentially as described by Vallee et al. [23, 24]. The modified albumin was dialyzed against water, for 4 h at 4 °. The extent of tyrosine nitration was determined by amino acid analysis, as described by Sokolovsky et al. [24]. (b) Modification with 2-nitrophenylsulfenyl chloride [25]. To 2 ml of a 0.35 albumin solution in 8 M urea/0.1 M sodium acetate buffer, pH 4.0, was gradually added 3.8 mg of sulfenyl chloride with effective stirring (note that the pH was kept above the optimum for the reaction, in order to avoid the N-F transition of albumin). The reaction was allowed to continue for 1 h, the pH being kept constant by addition of 0.02 M NaOH. The reaction was stopped by gel-filtration through a 1.2 x 40 cm column of Sephadex G-25, preequilibrated with the urea-acetate buffer solution. The modified albumin was eluted with 0.1 M sodium acetate buffer, pH 4.0, dialyzed against water for 12 h at 4 °C, and lyophilized. Modification of tryptophanyl residues was established by measuring absorbance at 365 nm [25], and by determination of unreacted tryptophan by the N-bromosuccinimide method [26]. Protein concentration was measured by dry weight. (c) Modification with glyoxal. The conditions were essentially those used by Nakaya et al. [27] for the reaction of lysozyme and the B chain of insulin with glyoxal. The unreacted arginyl and lysyl residues were measured by the N-hydroxyquinoline [28] and the trinitrobenzenesulfonic acid [29] methods, respectively. (d) Modification with acetic anhydride. The conditions were as used by Jonas and Weber [30]. The unreacted e-amino groups were measured by the ninhydryn method [31 ]. (e) Modification withformaldehyde. The conditions were as used by Jonas and Weber [30]. The unreacted e-amino groups were measured by the trinitrobenzenesulfonic acid method [29]. (f) Unfoldingand reduction of disulfide linkages. To 2 . 0 ~ albumin solution in 0.1 M Tris-HC1 buffer pH 8.0, urea was added to a concentration of 8.0 M; the pH was adjusted to 9.0, and 2-mercaptoethanol [32] was added to a concentration of 0.3 M (1/tl per mg of albumin). The solution was allowed to stand at room temperature (22-24 °C) for 4 h, under nitrogen. Free -SH groups in the reduced albumin were determined by the 5,5'-dithiobis(2-nitrobenzoic acid) method, as described by Habeeb [32]. (g) Oxidation of reduced albumin was performed as described by Andersson [33]. (h) S-carboxyamidomethylation of reduced albumin. To the denatured reduced albumin solution (a sample containing 100 mg albumin) was added 540 mg iodoacetamide [34] and left in the dark for 20 min under nitrogen. An aliquot of reaction mixture was exhaustively dialyzed against water at 4 °C (to remove urea and nonreacted iodoacetamide). Carboxamidomethylated cysteine residues were measured by total amino acid analysis [35]. The concentration of the modified albumin solution was measured using the procedure of Lowry et al. [36]. (i) S-carboxamidomethylation of native albumin was performed essentially as described by Nikkel and Foster [37]. Free sulfhydryl groups were measured by the 5,5'-dithiobis (2-nitrobenzoic acid) method, as described by Janatova et al. [38]. Total amino acid analysis was performed with a Technicon Aminoacid Analyzer

180 (Type I), as described by Spackman et al. [35]. Before analysis, the protein was hydrolyzed with 6 M HCI, for 20 h at 110 °C. RESULTS

Effect of the modification of amino acid residues on steroid-albumin interaction Fig. l(a) illustrates the titration of steroid binding affinity of albumin samples modified in tyrosyl residues (with tetranitromethane) or in one tryptophanyl residue (with 2-nitrophenylsulfenyl chloride). Fig. l(b) shows the result of a similar titration with albumin samples modified in the basic amino acid residues with glyoxal, formaldehyde or acetic anhydride. Albumin binding affinity was titrated with 11deoxycorticosterone and epitestosterone, respectively, using the equilibrium dialysis method. The results obtained are presented as double reciprocal plots in accordance with Eqn. 2, from which the binding parameters can be calculated. The corresponding values are presented in Table I. It is seen that with 11-deoxycorticosterone as titrant, amino acid modification significantly decreased affinity, as results from comparison of the binding constants (K) obtained with modified (M) and native (N) albumin, respectively. Thus, the K(M):K(N) ratio was 0.23 after modification of one tryptophanyl; 0.11 after modification of 6 (32 ~) tyrosyls; 0.17 after modification of 17 (74~) arginyls with glyoxal, and 0.37 after modification of 7 (30~) arginyls with formaldehyde. Treatment with glyoxal and formaldehyde affected a limited number of lysyls but the participation of these residues in binding must be of secondary importance, as indicated by the results obtained with the acetylated albumin (K(M):K(N) = 0.93 after modification of 13 lysyl residues). The steroid binding affinity of nitrated albumin was also titrated with epitestosterone, and a significant decrease of the binding constant value was observed, which, nevertheless, was smaller than with l l-deoxycorticosterone. In contrast with these results, modification of albumin free -SH group with iodoacetamide did not alter affinity for epitestosterone (data omitted). The binding constants of albumin samples modified in the basic amino acid residues were also determined by fluorimetric titration and the corresponding Kr values can be compared in Table I with those obtained by equilibrium dialysis (K values). The consistency of K and Kf values indicates that quenching of albumin fluorescence and steroid binding were equally affected by modification of amino acid residues. Table I also includes the AG corresponding to the steroid interaction with the different albumin samples. It is worth noting that after modification of one tryptophanyl residue, AG was less negative by about --1 kcal/mol, a value that fits in well with the A G of transfer of one tryptophanyl residue from water to a hydrophobic domain [39]. In order to establish whether the function of amino acid residues in steroid binding was dependent on the structure of the ligand, samples of albumin modified in the basic amino acid residues were assayed with the steroids listed in Table II. The results presented confirm the original hypothesis since the calculated K(M):K(N) ratios varied according to the steroid structure. The contribution of lysyl residues to steroid binding is demonstrated with testosterone acetate. Native albumin is a mixture of molecular species, namely mercaptalbumin, which has one free -SH group per mol, and non-mercaptalbumin, which has none [40].

181 I

I

I

I

I

I

I

I

I

j~

l

/

I

(a)

7 /NPS

E[

E-II

I

I

I

I

I

I

I

01

I

O2

i

i

i

i

i

(b)

5

4

l --

3

AA

2

I

I

I

I

I

01

I

I

I

I

I

0 2

Fig. 1. (a) Effect of the modification of tyrosyl and tryptophanyl residues on albumin affinity for steroids. Affinity measured by equilibrium dialysis; other experimental conditions were as described in Materials and Methods. The steroid concentration ([S]) is indicated in the abscissa. D (©, Q, ~ ) titration with 11-deoxycorticosterone; E (73, ll) same, with epitestosterone. TNM, albumin modified with tetranitromethane; NPS, albumin modified with 2-nitrophenylsulfenyl chloride; C, control (native) albumin. The number of modified amino acid residues is stated in Table I. (b) Effect of the modification of basic amino acid residues on albumin affinity for steroids. Affinity titrated with 11deoxycorticosterone. G, F and AA indicate pretreatment of albumin with glyoxal, formaldehyde and acetic anhydride, respectively; C, control (native) albumin. Other conditions were as described in (a) and in Materials and Methods.

182 TABLE I I N T E R A C T I O N OF I I - D E O X Y C O R T I C O S T E R O N E A N D EPITESTOSTERONE WITH NA. TIVE A N D M O D I F I E D A L B U M I N Experimental conditions were described in Materials and Methods. K and n (Eqn. 2) values wer~ determined by equilibrium dialysis, at 25 °C, while the K~ (Eqn. l) values (in parenthesis) were determined by fluorimetric titration, at 25 °C. Albumin concentration was 20/~M (A, equilibrium dialysis method), 15 # M (A, fluorimetric method) and 8/zM (B, fluorimetric method). Experiment

Modifier

Modified amino acid residues (per mol of albumin)"

n *~

K (or Kf) × l0 -4 ( M - 1)

fiG (kcal/mol)

None

2-NitrophenylTrp (1) sulfenyl chloride Tetranitromethane Tyr (6) Glyoxal Arg (17); Lys (7)

1.2 -1.2

15.3 (15.1) 3.5

-7.1 -7.1 --6.2

1.2 1.2

1.6 2.6

--5.7 --6.1

Formaldehyde

1.2

Steroid; l l-deoxycorticosterone A

None

--

Arg (7); Lys (13)

Acetic anhydride

Arg (0); Lys (13)

(3.6)

--6.1

5.6

-6.5

--

(7.0)

-6.5

1.2

14.3

--7.0

--

(14.8)

--7.0

Steroid; epitestosterone A B

None Tetranitromethane None lodoacetamide

None Tyr (6) None Cys-SH (0.6)

1.2 1.2 --

7.3 1.6 (8.2) (8.2)

--6.6 -5.7 -6.6 -6.6

* Average of values resulting from two independent measurements. The total number of residues (per mol of albumin) is 2 Trp, 19 Tyr, 23 Arg, 58 Lys [ref. 51], and 1 Cys (mercaptalbumin [37, 40]). *" Values calculated by the least squares method. TABLE II I N T E R A C T I O N OF STEROIDS WITH A L B U M I N SAMPLES M O D I F I E D IN THE BASIC A M I N O ACID RESIDUES Experimental conditions were as described under Materials and Methods and Table I. Fluorimetric titration of binding affinity; albumin concentration was 14/~M. Steroid

Kt × 10 -4 (M -I) Native albumin

Epitestosterone Androsterone Testosterone acetate 17fl-Hydroxy- 17a-methyl4,9( 11)androstadien-3-one 17a-Hydroxy- 11-deoxycorticosterone 21-acetate

Glyoxaltreated albumin

(a)

(b)

Formaldehydetreated albumin (c)

Acetic anhydridetreated albumin (d)

K ( M ) : K (N)* (b/a)

(c/a)

(d/a)

8.2 12.1 28.7

5.1 6.8 13.1

6.2 9.7 14.5

8.2 12.6 21.5

0.62 0.56 0.46

0.76 0.80 0.50

0.98 1.04 0.75

11.1

3.7

7.1

10.6

0.33

0.64

0.95

14.3

7.5

10.6

13.8

0.52

0.74

0.96

* K (M), affinity constants obtained with modified albumins ((b) -- (d)); K(N); as with native albumin (a).

183 Measurement of free -SH groups in native albumin gave 0.65 -SH per mol which means that the employed sample was largely constituted by mercaptalbumin. Mercaptalbumin and nonmercaptalbumin fractions were separated by chromatography on DEAE-Sephadex A-50, and representative samples of these components were assayed for binding to the steroids listed in Table II. Fluorimetric titration of affinity did not reveal significant differences between the assayed fractions (experimental data omitted), which leads one to conclude that the free -SH group of albumin was not involved in steroid binding. This conclusion is consistent with the negative effect of Scarboxamidomethylation in Table I.

Effect of steroids on the fluorescence of the system 1-anilinonaphthalene-8-sulfonatealbumin Since Weber and Laurence's initial observations [41], 1-anilinonaphthalene-8sulfonate is used as a fluorescent probe for hydrophobic sites of albumin. The dye emission can be excited by transfer of the excited state of albumin tryptophanyls [42, 43], and accordingly, the dye is a suitable detector of interactions in the neighborhood of albumin tryptophan. Table III allows one to compare the influence of several steroids on (a) 1-anilinonaphthalene-8-sulfonate fluorescence excited by transfer of energy from tryptophanyl residues (excitation at 285 nm; emission at 468 nm); (b) the intrinsically excited fluorescence of 1-anilinonaphthalene-8-sulfonate (excitation at 372; emission at 468 nm); (c) albumin intrinsic fluorescence in the presence of l-anilinonaphthalene-8-sulfonate (excitation at 285 nm; emission at 342 nm); and (d) albumin intrinsic fluorescence in the absence of 1-anilinonaphthalene-8sulfonate (excitation and emission as in (c)). Steroids were added dissolved in 0.1 M Tris. HC1 buffer, pH 8.0, in order to avoid the effect of ethanol on the dye fluorescence. The dye/albumin molar ratio was ~ 5 which, according to available data [42], must ensure almost complete saturation of albumin binding sites for 1-anilinonaphthalene8-sulfonate. Since under the given experimental conditions the quantum yield offluor-

TABLE III EFFECT OF STEROIDS ON THE FLUORESCENCE OF THE SYSTEM 1-ANILINONAPHTHALENE-8-SULFONATE-ALBUMIN Experimental conditions were as described in text and Materials and Methods. 8.0/zM albumin; 50/~M 1-anilinonaphthalene-8-sulfonate. Kf values were determined by fluorimetric titration, at the indicated wavelengths. Steroid

Kt × 10-4 (M -1) 1-Anilinonaphtalene-8-sulfonate-albumin

Albumin

285 ~ 468 (nm) 19-Nortestosterone 11.6 1,4-Androstadien-3,17-dione 14.2 Corticosterone 16.6 Prednisone 9.8

372 ~ 468 (nm)

285 ~ 342 (nm)

285 ~ 342 (nm)

-- * - * - * - *

11.8 13.1 16.5 8.8

11.2 13.6 17.3 9.3

Steroid addition did not modify albumin fluorescence beyond the limit of the experimental error.

184 escence of free 1-anilinonaphthalene-8-sulfonate was negligible and the dye was firmly bound to albumin [42], it can be assumed that quenching of 1-anilinonaphthalene-8-sulfonate emission in condition (a) was unequivocally dependent on steroid binding to albumin. Moreover, in conditions (a), (c) and (d) the assayed steroids quenched fluorescence with equal effectiveness, while in condition (b) the quenching effect was below the limit of experimental error. Since under the latter conditions steroids interacted with albumin as under condition (a), it may be concluded that steroids quenched l-anilinonaphthalene-8-sulfonate fluorescence only when it was excited by non-radiative energy transfer from the excited tryptophanyl residues of albumin.

Polarity of the steroid binding site Spectroscopic changes observed when chromophores are noncovalently bound to proteins have been used to evaluate the physical properties of binding sites [44]. Binding of A4-3-oxo steroids to albumin causes a shift to the blue of the steroid ultraviolet spectral maximum (J'max) [18] and similar shifts occur when steroids are transferred to apolar solvents [18]. Fig. 2 shows the 2m,x of several Aa-3-oxo steroids plotted as a function of Z, an empirical solvent polarity scale suggested by Kosower [45]. 2m~, of the steroids in albumin/Tris.HCl buffer is superimposed upon the standard line so that projection of the respective points on the abscissa axis shows the polarity of the steroid binding site. The results presented apparently indicate that most of the assayed steroids bind at a relatively apolar site (Z = 90). On the other hand, 250

i

I

I

I

i

I

I

I

~

I

I

I

248

246

.S t

I

i

i

I

I

i

f

244 E 242

Y

24C I~O 238 75

(85)

. . . . . . . . 80

(5O)

l,

,,, 85

,1, 90

, , ,

, 95

Z vglue

Fig. 2. Ultraviolet spectral maximum (2max) of steroids after binding to albumin; comparison with the respective 2ma. in media of known polarity. Steroids (15 I~M)dissolved in 3 ml of 32 #M albumin in 0.1 M Tris.HC1, pH 8.0; 1 cm optical path. The calibration curve was obtained with the same steroids dissolved in methanol, isopropanol- or ethanol-water mixtures, which Z values are stated in the abscissa. A (A, II, O), steroids in the albumin-Tris solution; /% D, O, steroids in standard solvents. TA, testosterone acetate; E, epitestosterone; MT, 17fl-hydroxy-17co-methyl-4,9(11)androstadien-3-one. The figures in parenthesis near the abscissa indicate the percentage (v/v) of ethanol in the ethanol-water mixture corresponding to the Z value indicated by the arrow.

185 the greater blue shift of testosterone acetate absorption in the albumin complex does not necessarily result from binding to a different site. A different orientation of the steroid to the hydrophobic residues at the same site may have the same effect.

Influence of p H on steroid binding Hydrogen ion concentration does not appreciably affect steroid solubility in aqueous protein-free solutions [46-48] and, therefore, the effect of pH variation on the steroid albumin-interaction is, in all probability, the consequence of the change in the ionization state of albumin side chains. Measurement of the pH influence on steroid binding was performed by the fluorimetric method and the results obtained are presented in Fig. 3. It is seen that the association constant of the assayed steroids increased continuously when the pH was raised from 5.0 to 9.5 (the pH of maximum interaction), while from 9.5 to more alkaline pHs, affinity decreased rather steeply (Fig. 3(a)). The effect of the pH varied somewhat according to the steroid structures, as illustrated in Fig. 3(b), where the modification of binding affinity as a function of pH is represented by the corresponding 6AG. It may be observed that the shift of pH from 5.0 to 9.5 increased 6AG by about --1.3 kcal/mol with cortisol, but only by about --0.4 kcal/mol with the other steroids. Since tyrosyl residues in proteins have a pK~ of 9.5 [49], the inflection at pH 9.5 indicates the participation of protonated tyrosyl residues in steroid binding, as earlier suggested by Schellman et al. [47], and Levedahl and Perlutter [48] on the basis of direct titration of steroid binding. Influence of albumin denaturation on steroid binding As most proteins with an ordered native structure, albumin undergoes a marked transition to the random coil state upon addition of guanidine.HCl and urea [39]. Fig. 4 shows that the modification of albumin structure determined a significant diminution of affinity for testosterone acetate, and moreover, with 4 M guanidine.HCl the interaction was practically suppressed. The effects of guanidine.HCl and urea correlate well with their potency as protein denaturants, the modification of albumin folding state being revealed by (a) the shift to the red of the fluorescence emission spectrum (Fig. 4, inset); (b) the decrease of the quantum yield of albumin fluorescence (Fig. 4, inset); and (c) the decreased ellipticity of albumin solutions (Fig. 4). The necessity of a folded polypeptide chain for steroid binding was further confirmed in conditions excluding an effect of denaturants on steroid solubility. The experimental procedure employed may be summarized as follows. Albumin was treated with urea and 2-mercaptoethanol (details as described in Materials and Methods) and samples of modified albumin were used for (a) titration of free -SH groups with 5,5'-dithiobis(dinitrobenzoic acid) [32]; (b) treatment with iodoacetamide, exhaustive dialysis and total amino acid analysis [35]; and (c) dilution and reoxidation of the unfolded albumin as described by Andersson [33]. Finally, samples of native and refolded albumin were fractionated by gel-filtration on Sephadex G-150 in or der to isolate the corresponding monomers. The steroid binding affinity of the different proteins was fluorimetrically titrated and the resulting Kf values are presented in Table IV. It is seen that: (a) treatment with urea and 2-mercaptoethanol determined reduction of nearly all of albumin disulfide bridges, which should involve a complete transition of the folded structure to the random coil state [39]; (b) denaturation, reduction and S-carboxamidomethylation of free -SH groups decreased the steroid

186 161

201

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16

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IF-

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5

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6

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/

~

:

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Fig. 3. (a) Influence of pH on the association constant (Kf) of the steroid-albumin complexes. Fluorimetric titration of affinity; 13 #M albumin; pH as indicated in the abscissa. The buffers used were (0.1 M): sodium acetate (pH 5-6); phosphates (pH 6-8); Tris-HC1 (pH 8-10); NH4CI]NH4OH (pH 10-11). Other conditions were as described in Materials and Methods. E, epitestosterone; MT, 17fl-hydroxy-17a-methyl-4,9(ll)androstadien-3-one; A, androsterone; C, cortisol; Pd, prednisone; Pt, 4-pregnene-3,11,20-trione. (b) Influence of pH on the AG of steroid-albumin interaction. 8AG is the difference between the AG at the pH indicated in the abscissa and the AG at pH 5. E, TA, Pt and C as in (a). b i n d i n g affinity below the limit of the experimental error; a n d (c) elimination of p e r t u r b a n t a n d slow reoxidation of the denatured protein re-established the steroida l b u m i n interaction to its n o r m a l strength. It must be pointed out that the fluorescence

187 3(

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Fig. 4. Influence of urea (U) and guanidine. HCI (G) on the formation constant (Kr) of the testosterone acetate-albumin complex. Fluorimetric titration of affinity. 13 # M albumin; standard experimental conditions, except perturbant which concentration is indicated in the abscissa. The figures in parenthesis near the abscissa indicate the percentage of a-helix structures in albumin. These values were calculated with the following [012~: --19,750 (native albumin); --9800 (albumin in the presence of 6 M urea); --3,300 (albumin in the presence of 4 M guanidine. HCl). Inset. Emission spectrum of albumin in the presence of guanidine. HC1. 15 ffM albumin in 0.1 M Tris. HCI, pH 8.0; excitation at 285 nm. The figures in parenthesis indicate the perturbant molarity. Other conditions were as described in Materials and Methods. TABLE IV STEROID BINDING TO NATIVE, U N F O L D E D AND REFOLDED ALBUMIN The experimental conditions were as described in Materials and Methods and text. Fluorimetric tritration of affinity; albumin concentration was 8.0 ffM. Condition

Treatment of albumin

Albumin state

Number of Cys per tool of protein

(A) (B)

None Fractionation *

(C)

Urea; 2-mercaptoethanol; iodoacetamide Urea; 2-mercaptoRefolded 0.6 ethanol; oxygen** Same as (D); fracRefolded -tionation" (monomer)

(D) (E)

Folded 0.6 Folded -(monomer) Unfolded 34.0"**

Kr x 10 - 4 ( M -1) Testosterone 17a-Hydroxy-11acetate deoxycorticosterone 21-acetate 29.3 29.9

16.3 18.0

-- . . . .

-- . . . .

28.6

18.7

30.8

18.0

" Fractionation by gel-filtration on Sephadex G-150 (2.8 x 100 cm column); 0.1 M Tris.HCl buffer pH 8.0 as eluant; temp. 4 °C [33]. ** The reduced albumin solution was slowly diluted with 0.1 M Tris.HCl buffer, pH 8.0, to a protein concentration of about 0.3 mg/ml and then allowed to stand for 4 days with free access to air. The solution was concentrated by low pressure dialysis [33]. *** Average of direct measurement of Cys (34.5 -SH) and total amino acid analysis of iodoacetamide-treated albumin (33.2 carboxamidomethylcysteine). Note that the number of disulfide bridges in native albumin is 17-18 [51]. . . . . Steroid addition did not modify albumin fluorescence beyond the limits of the experimental error,

188 spectrum and the elution pattern on Sephadex G- 150 of the refolded albumin monomer could not be distinguished from those of the native monomer (results omitted).

Influence of temperature on steroid binding Fluorimetric titration of steroid-albumin interaction shows that increasing the temperature in the 9-37 °C range decreased the association constant value (Fig. 5) The data in Fig. 5 allow one to calculate the thermodynamic parameters AG, A H and I

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Fig. 5. Influence of temperature on steroid-albumin interaction. Fluorimetric titration of affinity. Albumin concentration was 15 #M, except with epitestosterone, that was 10 ffM. Standard experimental conditions, except temperature, that is indicated in the abscissa. TA, testosterone acetate; Pt, 4-pregnene-3,11,20-trione;D, 11-deoxy-17a-hydroxycorticosterone21-acetate; E, epitestosterone; C, cortisol. d S and the corresponding values are presented in Table V. It is seen that the interaction involved (a) a negative free (Gibbs) energy change, indicating the spontaneous formation of the steroid-albumin complex; (b) a small negative enthalpy change, indicating that the interaction itself was favored at the higher temperatures so that the heat content of the system decreased; and (c) a relatively large positive entropy change, that with some steroids accounted for 60-70 ~ of the free energy change. The increase of entropy followed the polarity rule, since it was relatively less with the more polar steroids. The general pattern of the temperature effect in Table V is consistent with that reported by other workers [3, 10, 47] using non-fluorimetric methods.

Fluorescence polarization measurements In standard experimental conditions (15 ffM albumin) addition of progesterone, in the concentration range of 50-200 # M did not modify the polarization of albumin intrinsic fluorescence (experimental data omitted).

189 TABLE V THERMODYNAMIC PARAMETERS OF STEROID-ALBUMIN COMPLEXES. FLUORIMETRIC TITRATION The experimental conditions were those as described in Fig. 5. The integration for the enthalpy change (AH) was made over the whole temperature range studied. Kf at 9 °C, 23 °C and 37 °C ( × 10-4 (M-I)): 22.0, 19.0 and 15.0 (17a-hydroxy-6a-methyl-4-pregnene-3,20-dione); 14.0, 11.0 and 8.0 ( 17fl-hydroxy-17a-methyl-4,9(11)androstadien-3-one); other values are taken from Fig. 5. AG values calculated from Kr values at 23 °C. Steroid

AG

2H

TAS

AS

(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) (cal/°g) Testosterone acetate 17a-Hydroxy-6a-methyl-4-pregnene-3,20-dione 4-Pregnene-3,11,20-trione 17a-Hydroxy-ll-deoxycorticosterone-21-acetate 17fl-Hydroxy-17a-methyl-4,9(1l)-androstadien3-one Epitestosterone Cortisol

-- 7.4 -- 7.1 -- 7.1 --7.0

-- 1.8 -- 2.3 - 2.4 --4.2

5.6 4.8 4.7 2.8

+ 19 + 16 + 16 + 9.5

--6.8 --6.7 --6.5

--3.3 --4.4 --5.2

3.5 2.3 1.3

+ 12 +7.8 +4.4

DISCUSSION Examination of steroid structures that determine binding to albumin leads one to postulate that the interaction is essentially due to hydrophobic bonding that results, on the one hand, from the inability of the steroid hydrocarbon framework to form hydrogen bonds with water, and on the other, from the accessibility of hydrophobic amino acid residues to a relatively apolar binding site in albumin [5, 6]. The site should be relatively small, since the area covered by typical steroid, such as estradiol-17fl, does not exceed 100 A 2 [50]. The lack of a strict specificity in the steroid-albumin system (a "low-affinity" system [4]) might seem to preclude any rigid structural requirements in albumin to interact with steroids, but the available evidence (including the observations here described) confirms that specific amino acid residues play a significant role in steroid binding. In that context, tryptophan residues (2 residues per mol of albumin [51]) must be considered first, because the indole group is primarily responsible for albumin fluorescence [15], and accordingly, quenching of fluorescence indicates that steroids interact near enough to the tryptophanyl residues so as to change the environment of the latter. This hypothesis is supported by (a), the decreased binding affinity of albumin after modification of one tryptophanyl residue (Fig. l(a) and Table I), and (b), steroid interference with 1-anilinonaphthalene-8-sulfonate excitation from the excited indole fluorophore (Table III). The function of a tryptophanyl residue in steroid binding agrees well with steroid interaction with L-tryptophan in aqueous solutions [8] and with steroid quenching of L-tryptophan fluorescence [9]. It is worth noting that after modification of one tryptophanyl residue, the L/G of the steroidalbumin interaction was less negative by --0.8 kcal/mol (Table I) a value that approaches both the AG of the steroid-L-tryptophan interaction (--1.0 kcal/mol) [8, 9] and the A G of transfer of one tryptophanyl residue from water to an apolar domain

190 [39]. Since the albumin molecule contains two tryptophans, at least one of these must be involved in steroid binding, in all probability the residue present in peptide sequence P Ic isolated from citraconylated albumin by Jonas and Weber [52]. Tyrosyl residues may also contribute to binding since nitration of 30 9/0tyrosyls significantly decreased affinity (Fig. l(a) and Table I) and the same occurred after deprotonation of the tyrosyl phenol (Fig. 3). The effect of nitration is consistent with: (a) that of ketonization of albumin tyrosyl residues [10]; (b) steroid interaction with L-tyrosine [8]; (c) steroid quenching of L-tyrosine fluorescence [9]; (d) steroid perturbation of tyrosine chromophores in albumin [7]; and (e) steroid binding to the tyrosine rich peptide sequence isolated from albumin by Pearlman and Fong [53]. Modification with formaldehyde and glyoxal, which affected arginyl residues, markedly decreased albumin affinity for steroids (Fig. l(b), and Tables I and II). Since acetylation of lysyl residues alone had no significant effect on binding (except with testosterone acetate) (Table II) it appears that in the formaldehyde and glyoxal derivatives it was the modification of the arginyl residues that caused the decrease of affinity for steroids. The effectiveness of the smaller hydrocarbon portions of arginine side chain as hydrophobic moiety is considered to be negligibly small [39], and consequently, the contribution of the arginyl residues must be determined by the protonated guanidinyl (pKa of 12.5 [49]), that is a good proton donor for hydrogen bonding with steroid carbonyl groups. On the other hand, acetylation of lysyl residues decreased albumin affinity for testosterone acetate, an effect that contrasts with the apparent ineffectiveness of these residues for the binding of the other steroids listed in Tables I and II. The contribution of the hydrocarbon portion of the lysyl side chain for binding may be estimated as being about -- 1.0 kcal/mol [39], and therefore, lysine participation in steroid binding may be nonpolar, in good agreement with the relatively higher hydrophobicity of the testosterone acetate binding site in Fig. 2. The peculiar behaviour of testosterone acetate, in Table II and Fig. 2, as well as a different effect of pH on steroid binding in Fig. 3, strongly support the view that steroids interact at somewhat different sites in albumin, according to the structural characteristics of the steroid molecule. A similar reasoning is valid for the variation of albumin affinity for 11-deoxycorticosterone and epitestosterone, after treatment with tetranitromethane (Table I). According to Jonas and Weber [30], changes in the fluorescence spectrum of the formaldehyde treated albumin indicate the proximity of at least one tryptophanyl residue to the basic amino acid residues. Since human serum albumin, that binds steroids very much like bovine serum albumin, also includes a sequence in which a tryptophanyl residue is in the proximity of basic amino acid residues [54], juxtaposition of apolar residues with cationic ones at the surface of albumin appears to be the molecular basis for steroid binding. This assumption fits in well with (a) the accessibility of aromatic residues to the albumin surface [55]; and (b) the decreased strength of the steroid albumin interaction after conversion of the aromatic apolar residues into their more polar derivatives, as exemplified by the 2-nitrophenylsulfenylation of one tryptophanyl residue, and the nitration (or protonation) or tyrosyl residues (Fig. l(a); Table I, and Fig. 3). The relative apolarity of the steroid binding site, as expressed on the Kosower scale (Fig. 2), may well reflect the presence of both apolar (Trp, Tyr and Lys (deprotonated)) and apolar (Arg and Lys (protonated)) residues. In this context, it seems pertinent to recall the early suggestion by Schellman et al.

191 [47] (supported by the effect of temperature in Fig. 5 and Table V), that steroids break (or bridge) ammonium phenolic crosslinks in albumin, to reduce local strain and thus increase entropy with very small enthalpy change. The conformational change resulting from the interaction must be, nevertheless, limited, as indicated by the negative results of the fluorescence polarization measurements. Native albumin exists as a single polypeptide chain possessing about 50~ helical structure, the protein folded state being stabilized by 17-18 disulfide bridges [51 ]. This molecular configuration is essential for steroid binding, as indicated by the effect of guanidine •HC1 and urea in Fig. 4, and those of the same perturbants plus 2-mercaptoethanol, in Table IV. In fact, with intact disulfide bridges (Fig. 4), the perturbants determine a transition of the folded polypeptide chain to the random coil state (cross-linked random coils) in which non-covalent interactions are disrupted and hydrophobic amino acid residues gain the greatest free energy by transfer to the perturbant solution [39]. These changes, that are evidenced by the decreased ellipticity of albumin solutions and by the shift to the red of albumin tryptophanyl emission (Fig. 4), may explain the decreased binding affinity of denatured albumin. In this connection is may be recalled that a similar decrease of affinity was observed by Attallah and Lata [9] with urea denatured albumin and testosterone, but they attributed the effect of urea to (a) solubilization of testosterone; (b) complete displacement of the steroid molecule; (c) an alteration of the hydration state of the protein, or (d) a combination of these. Although the mechanism quoted should not be ignored in order to explain the effect of guanidine. HC1 and urea in Fig. 4, the possibility of steroid solubilization by denaturants is minimized by the absence of a 17-OH group in testosterone acetate (a prerequisite for steroid interaction with urea [56]). On the other hand, it is seen in Fig. 4 that the effect of denaturants correlates well with their action on albumin folding state, a circumstance that strengthens the conviction that the denaturants affected steroid binding by unfolding of the polypeptide chain and exposure of hydrophobic groups to the solvent. The requirement of the normal configuration of the polypeptide chain for steroid binding is unequivocally confirmed by the effect of reduction and S-carboxamidomethylation (Table IV), a modification that ensures a complete transition of albumin folded state to the random flight conformation, under experimental conditions excluding a direct effect of urea on steroid binding. Fluorescence quenching titration has been recommended by Attallah and Lata [9] as a sensitive method for the study of steroid-albumin molecular interactions. In contrast to other methods, such as equilibrium dialysis, spectrophotometry, etc, fluorimetric titration has seldom been used. The present study shows that measurement of steroid binding parameters by fluorimetric titration and equilibrium dialysis yields consistent values, and furthermore, the patterns of affinity variation as a function of pH and temperature obtained by fluorimetric titration, agree well with those reported by other workers [3, 10, 47, 48] using nonfluorimetric methods. Our results demonstrate that steroid binding and quenching of tryptophan emission are strictly related phenomena, thus confirming the validity of the fluorescence titration method. ACKNOWLEDGEMENTS This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Tdcnicas, Argentina, The Jane Coffin Childs Fund for Medical Research,

192 a n d The Scientific Office, A m e r i c a n States Organization. Professor Gregorio Weber c o n t r i b u t e d with his criticism a n d c o m m e n t s to the typescript preparation. Technical advice a n d instruments for protein analysis were kindly provided by the staff of D e p a r t a m e n t o de Q u i m i c a Biol6gica, F a c u l t a d de Bioquimica y Farmacia, in particular by Professor A. C. Paladini, Dr. Mirta Biscoglio a n d Dr. Silvia Daurat. Calculation of b i n d i n g constants a n d free energy values was performed by Miss Silvia H a r t m a n n (Centro de C o m p u t a c i 6 n , F a c u l t a d de Medicina). A . M . R . is a visiting research fellow, Servicio de Cooperaci6n Social, Ministerio del Trabajo, Spain (19711973). A.O.M.S. is a Career Investigator, Consejo Nacional de Investigaciones Cientificas y T6cnicas ( C O N I C E T ) , Argentina. REFERENCES 1 Westphal, U. (1961) in Mechanism of Action of Steroid Hormones (ViUee, C. A. and Engel, L. L., eds.), pp. 33-83, The MacMillan Co., New York 2 Markus, G., MacClintock, D. K. and Castellani, B. A. (1967) J. Biol. Chem. 242, 4402-4408 3 Alfsen, A. (1963) Compt. Rend. Trav. Lab. Carlsberg. 33, 415-431 4 Ryan, M. T. (1973) Biochemistry 12, 2221-2230 5 Westphal, U. (1971) Steroid-Protein Interactions, pp. 133-156, Springer Verlag, Berlin 6 Margineda, A,, Martino, E. and Stoppani, A. O. M. (1975) Biochim. Biophys. Acta 409, 376-386 7 Ryan, M. T. and Gibbs, G. (1970) Arch. Biochem. Biophys. 136, 65-72 8 Abelson, D., Depatie, C. and Cradock, V. (1960) Arch. Biochem. Biophys. 91, 71-74 9 Attallah, N. A. and Lata, G. F. (1968) Biochim. Biophys. Acta 168, 321-333 10 Oyakawa, E. K. and Levedahl, B. H. (1958) Arch. Biochem. Biophys. 74, 17-23 11 Yielding, K. L. and Tomkins, G. M. (1960) Proc. Natl. Acad. Sci. U.S. 46, 1483-1488 12 Ranieri, R. and Levy, H. R. (1970) Biochemistry 9, 2233-2243 13 Stoppani, A. O. M., Brignone, C. M. C. and Brignone, J. A. (1968) Arch. Biochem. Biophys. 127, 463-475 14 Baulieu, E. E. (1975) J. Mol. Cell. Biochem. 7, 157-174 15 Teale, F. W. J. (1960) Biochem. J. 76, 381-388 16 Leonard, Jr. W. J., Vijai, K. K. and Foster, J. F. (1963) J. Biol. Chem. 238, 1984-1988 17 Ryan, M. T. (1968) Arch. Biochem. Biophys. 126, 407-417 18 Westphal, U. (1957) Arch. Biochem. Biophys. 66, 71-90 19 Velick, S. F. (1958) J. Biol. Chem. 233, 1455-1467 20 Westphal, U., Ashley, B. D. and Selden, G. L. (1958) J. Am. Chem. Soc. 80, 5135-5138 21 Klotz, I. M., Walker, F. M. and Pivan, R. B. (1946) J. Am. Chem. Soc. 68, 1486-1490 22 Bewley, T. A., Brovetto-Cruz, J. and Li, C. H. (1969) Biochemistry 8, 4701-4708 23 Riordan, J. F. and Vallee, B. L. (1972) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N., eds.), Vol. 25B, pp. 515-521, Academic Press, New York 24 Sokolovsky, M., Riordan, J. F. and Vallee, B. L. (1966) Biochemistry 5, 3582-3589 25 Scoffone, E., Fontana, A. and Rocchi, R. (1968) Biochemistry 7, 971-979 26 Patchornik, A., Lawson, W. B., Gross, E. and Witkop, B. (1960) J. Am. Chem. Soc. 82, 59235927 27 Nakaya, K., Horinishi, H. and Shibata, K. (1967) J. Biochem. 61, 345-351 28 Ceriotti, G. and Spandrio, L. (1957) Biochem. J. 66, 607-610 29 Habeeb, A. F. S. A. (1966) Anal. Biochem. 14, 328-336 30 Jonas, A. and Weber, G. (1971) Biochemistry 10, 1335-1339 31 Harding, V. J. and MacLean, R. M. (1916) J. Biol. Chem. 24, 503-517 32 Habeeb, A. F. S. A. (1972) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N. eds.) Vol. 25B pp. 457-464. Academic Press. New York 33 Andersson, L.-O. (1969) Arch. Biochem. Biophys. 133, 277-285 34 Konigsberg, W. in Methods in Enzymology (Hirs, C .H.W. and Timasheff, S. N., eds.), Vol. 25B, pp. 185-188. Academic Press. New York 35 Spackman D. H., Stein, W. H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206

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