THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 13, Issue of July 10, PP. 4706-4712, 1973

Printed in U.S.A.

Polymorphism of Rhesus Monkey Serum Prealbumin

MOLECULAR PROPERTIES AND BINDING OF THYROXINE AND RETINOL-BINDING PROTEIN

(Received for publication, January 29, 1973)

PIETER P. VAN JAARSVELD,* WILLIAM T. BRANCH, HAROLD EDELHOCH, AND JACOB ROBBINS

Clinical Endocrinology Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 2001~

SUMMARY

Homogeneous preparations of the two homozygous types of rhesus monkey serum prealbumin, Pt l-l and Pt 2-2, were studied and compared with human prealbumin. Circular dichroism showed similar secondary structure, with a roughly equal distribution of /3 and unordered peptide groups. Minor differences between the two monkey proteins were observed in ultraviolet absorption and fluorescence of the tyrosyl and tryptophanyl residues between pH 4 and 2 and in the fluores- cence of 5-dimethylaminonaphthalene-1-sulfonate between pH 7 and 3. The changes were similar to those seen with human prealbumin although there were small but significant differences.

The behavior of Pt l-l and Pt 2-2 in concentrated solutions of guanidine revealed the remarkable stability of these pro- teins. The denaturation of monkey prealbumin is incom- plete in 7.3 M guanidine, pH 8.0, but goes to completion when the pH is reduced to values below pH 4.0. In contrast, denaturation of human prealbumin is complete in 7.3 M guanidine, pH 8.0.

Pt l-l, Pt 2-2, and human prealbumin showed similar binding of thyroxine (K,,,,, * lo* M-‘, R = 1) and retinol- binding protein (K,,,,, N lo6 M-l, n = 4), indicating simi- larity in their “active” sites.

The genetic polymorphism of serum prealbumin of rhesus monkeys (Mucacca mulatta) is inherited by autosomal co-domi- nance from two alleles (1, 2). The two homozygous types (Pt l-l and Pt 2-2) migrate as single sharp bands on polyacrylamide disc gel electrophoresis at pH 8.6. The hybrid type (Pt l-2) consists of five electrophoretic bands: the fast Pt l-l, the slow Pt 2-2, and three intermediate bands which represent the isomers formed by hybridization of the two homozygous tetramers (1,2).

Human prealbumin is similarly a tetramer of identical subunits (3-7) and also binds both thyroxine (8-12) and retinal-binding protein (13-16). The binding of RBPl to prealbumin serves to

* Recipient of a Public Health Service Tnternational Postdoc- toral Research Fellowship (FO5TWO1762). Permanent address, Department of Pharmacology, University of Stellenbosch, P. 0: Box 53. Bellville. Renublic of South Africa.

r The abbreviations used are: RBP, retinol-binding protein;

prevent rapid glomerular filtration (15, 17) since RBP has a molecular weight of 21,000 (13, 18). X-ray crystallographic analysis of human prealbumin (3) revealed the existence of a channel through the center of the tetrahedrally arranged sub- units in which a single thyroxine-binding site is presumably located. There are also four independent RBP-binding sites on prealbumin (16). The two types of binding sites are located in different places since bound thyroxine and RBP are inde- pendent of each other (14-16).

Human prealbumin is an unusually stable protein since it does not dissociate in either SDS or concentrations of guanidine HCl approaching 6 M (15, 19). The unusual binding properties of human prealbumin and its remarkable stability have prompted this study with the two homozygous forms of monkey pre- albumin. There are a few differences in the primary structures of human and monkey prealbumin as revealed by amino acid analysis, peptide mapping, and sequence studies2 Even fewer differences exist between Pt l-l and Pt 2-2.

MATERIALS AND METHODS

Protein Preparations-Pt l-l and Pt 2-2 rhesus prealbumin were purified from serum by chromatography on DEAE-Sepha- dex A-50 and preparative polyacrylamide gel electrophoresis. Details of the methods will be presented elsewhere.2 Lyophi- lized human prealbumin was purchased from Behringwerke and further purified by preparative polyacrylamide gel electrophoresis (5). Human retinol-binding protein was a gift from Dr. Dewitt S. Goodman and was purified as described previously (11, 13, 14). Protein concentration was determined by absorption at 280 nm using Efy,,, values of 14.1 and 16.8 for prealbumin and retinol- binding protein, respectively (11, 16).

Hybridization-Pt l-l and Pt 2-2 were dissociated by incubat- ing them separately at room temperature in 6 M guanidine HCl at pH 6 for 16 hours. Equal amounts of the two proteins were then mixed and the guanidine was removed by dialysis for 4 hours against 0.05 M phosphate buffer-O.1 M KCI, pH 7.4. The solution was then examined by polyacrylamide disc gel elec- trophoresis as described previously (2).

Circular Dichroism-The Cary model 60 spectropolarimeter equipped with a Pockels cell was used to measure circular di- chroism. Optical densities were between 1 and 2 at the experi-

Dns, 5dimethylaminonaphthalene-1-sulfonyl; SDS, sodium do- decyl sulfate; CD, circular dichroism.

$P. P. van Jaarsveld, manuscript in preparation.

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mental wave lengths. Two- and 0.2.cm cells were used in the near and far ultraviolet, respectively.

Ultraviolet Spectroscopy-Ultraviolet difference spectra were obtained at acid pH values and room temperature by the tandem cell technique (20) using a Cary model 14 spectrophotometer. Spectra were obtained with -0.3 mg per ml prealbumin solutions.

Ionization of tyrosyl groups at alkaline pH values was followed by the increase in absorbance at 295 nm. Solutions containing 0.5 mg per ml of prealbumin (0.05 M phosphate-O.1 M KCI, pH 7.4) were titrated to pH 13. A molar increase of 2400 was used to estimate the number of ionized phenolic groups (21).

Fluorescence-A Turner 210 spectrofluorometer was used to obtain fluorescence spectra and intensities at 25”. The ab- sorbance of all solutions was less than 0.05 at the excitation wave length. Tyrosyl and tryptophanyl emission were measured at 305 and 340 nm, respectively, by exciting at 280 and 295 nm. The temperature of the prealbumin solutions was maintained at 25”.

Preparation of Fluorescence Conjugates-Small volumes (1 to 2 ~1) of 5-dimethylaminonaphthalene-l-sulfonyl chloride in acetone were added to 0.05% solutions of prealbumin in 0.1 M

sodium bicarbonate (pH 8.3), the weight ratio of dye to protein being about 1%. The reaction was allowed to proceed for 4 hours at 4”. Unconjugated dye was removed from the protein by chromatography on Sephadex G-25 and then by extensive dialysis against a large volume of pH 7.4 solution containing 0.05 M phosphate-O.1 M KCl. Using a molar extinction coeffi- cient of 4200 (at 340 nm) for Dns (22) it was estimated that about 2 moles of dye were bound per mole of monkey prealbumin.

Polarization of Fluorescence-A modified Phoenix light scatter- ing photometer was used to measure the polarization of fluores- cence (23). Corning filters 5970 and 3385 with maximal trans- mission at 365 and 540 nm, respectively, were used in front of the incident and fluorescent beams to measure Dns fluorescence. The polarization of retinol fluorescence (bound to retinol-binding protein) was measured at 480 nm by exciting at 330 nm with unpolarized light using Corning filters 3387 and 7-54, respec- tively (16).

The polarization (P) was calculated from the intensities of the vertical (Iv) and horizontal (1,) components of the fluorescent light, i.e. Iv - IH/Iv + la. The relaxation time (ph) is obtained from the Perrin equation (22),

(++;) = ($+;)(1+;)

for unpolarized light. PO is the limiting value of P when T/q is zero and 7 is the fluorescence lifetime. ph of Dns conjugates of prealbumin was calculated from the slopes and intercepts of plots of l/P + 45 versus T/v, i.e. ptjO = (intercept/slope)

250 37(~/T)~~o. The relaxation time of a rigid, unhydrated sphere of the same mass (pO) may be calculated as 3rV/RT, where the hydrodynamic volume V = MC. For a molecule with a molecu- lar weight (M) of 54,000 and a partial specific volume (ti) of 0.73, py . IS 4.22 x lo-* s.

Fluorescence lifetimes (7) of Dns conjugates were measured with a modified nanosecond fluorescence decay time apparatus (TRW, Inc., El Segundo, California) as described by Chen (24).

Denaturation in Guanidine-The negative peak which devel- oped at 285 nm in t,he difference spectrum of prealbumin was used to follow the kinetics of denaturation in guanidine HCI solutions in a Cary model 14 spect.rophotometer. The initial absorbance in guanidine HCl was estimated by extrapolating the readings from the first 30 s to zero time. The total change in

absorbance was obtained by lowering the pH to 2.0 with HCl where the absorbance decreased immediately to the final value.

Binding of Thyroxine and Retinal-binding Protein-Equilibrium dialysis was used to measure the affinity of rhesus prealbumin for thyroxine (11). Prealbumin solutions (0.79 pM) in 1.5 ml of 0.05 M phosphate-O.1 M KCl-1 mM EDTA (pH 7.4) were dialyzed against 5 ml of the same buffer. Stock solutions of n-thyroxine, which contained n-[rZ51]thyroxine (Abbott Labora- tories, Chicago, Ill.), were prepared in 0.01 Y KOH and their concentrations measured by absorbance at 325 nm using an e = 6180 (25). Concentrations ranging from 0.1 to 1.0 pM were placed inside or outside the bags (Visking tubing) at the be- ginning of the dialysis. The binding data were analyzed accord- ing to Scatchard (26), i.e. v/C = Kn - KF where ij is the number of moles of thyroxine bound per mole of protein, n is the number of binding sites of thyroxine on prealbumin, C is the molar con- centration of free thyroxine, and K is the apparent association constant. The method of Sterling and Brenner (27) was used to correct for any iodide present at the time of analysis.

The binding of human RBP to rhesus prealbumin was studied by the polarization of fluorescence method as described earlier (16). The increase in polarization of RBP with prealbumin binding was analyzed by assuming that the two limiting polariza- tion values, i.e. in the absence of prealbumin and in an excess of prealbumin, represent the values for free and bound RBP and that complexes with more than one RBP have the same polariza- tion values as the one to one complex. The association constants were obtained by a curve fitting program developed by Dr. H. Saroff (National Institutes of Health).

RESULTS

Quaternary Xtructure

The molecular weights of Pt l-l and Pt 2-2, as determined by sedimentation equilibrium,2 were found to be similar to that of human prealbumin, i.e. about 54,000 (3, 5). The polyacryl- amide gel electrophoresis patterns of the two forms of monkey prealbumin are illustrated in Fig. 1. Hybridization of the two forms requires several days of incubation in aqueous media (2). Complete hybridization could be accomplished much faster by first dissociating the two proteins in 6 M guanidine HCI at pH 6, mixing, and then dialyzing for 4 hours against 0.05 M phosphate- 0.1 M KC1 buffer at pH 7.4. It has been shown recently that human prealbumin is stable in 5.7 M guanidine HCl at pH 8.0 but dissociates rapidly if the pH is lowered (19). The elec- trophoretic pattern obtained after this treatment is also shown in Fig. 1. The hybrid bands are equally displaced from each other with the center band having the highest relative staining intensity. Random recombination of the subunits should result in relative intensities of 1, 4, 6, 4, 1 for the five bands (2). The data are in approximate agreement with the expected results.

Secondary Structure

The CD spectra of both Pt l-l and Pt 2-2 in the near ultra- violet were very similar and resembled closely that reported for human prealbumin (5). Two well defined peaks at 290 and 283 nm, due to tryptophan, were present with molecular ellipticity values of $65,000 and +30,000, respectively.

The CD spectra of the two monkey prot.eins in the far ultra- violet region (240 to 200 nm) were also similar and close to that of human prealbumin. They contained a single negative extremum at 214 nm which, according to the previous analysis (5), can be attributed to a roughly equal distribution of /3 and unordered peptide groups.

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Pt I-1 HYBRID Pt 2-2

f-1

1-t)

FIG. 1. Polyacrylemide (8.5%) disc gel electrophoresis of Pt l-l, Pt 2-2, and hybrids of the two proteins. The hybrids were formed by incubating equal amounts of the two proteins in 6 M

guanidine HCl (pH 6.0) followed by dialysis for 4 hours against 0.05 M phosphate-O.1 M KC1 (pH 7.4). The faint band which moves more slowly than the native hybrid proteins is usually seen after dissociation in guanidine and is presumably due to aggrega- tion.

Tertiary Xtructure

Ultraviolet Absorption-Only trivial changes occurred in the near ultraviolet absorption of either protein between pH 7.3 and 4.0. Blue-shifted difference spectra with peaks centered near 290, 284, and 278 nm developed between pH 4 and 2 in both proteins (Fig. 2). The longest wave length peak represents perturbations of the tryptophanyl chromophores, while the peaks at 284 and 278 nm represent contributions from the tyrosyl as well as from the tryptophanyl residues (28). The pH depend- ence of the transition is shown in Fig. 3. There is a small but significant difference in the relative intensity of the tyrosyl and tryptophanyl difference peaks in the two polymorphic forms of prealbumin.

Phenolic lonizcltion-The ionization of the tyrosyl residues in Pt l-l and Pt 2-2 in 0.05 M phosphate-O.1 M KC1 is shown in Fig. 4. No time effects were observed during the spectrophoto- merit titration, and the final absorption values at pH 13.0

1 2io 280 290 300 310

WAVELENGTH (nm)

FIG. 2. Effect of pH on the ultraviolet difference absorption spectra. The reference solutions were Pt l-l (0.35 mg per ml) and Pt 2-2 (0.4 mg per ml) in 0.05 M phosphate-O.1 M KC1 (pH 7.3). Temperature = 25”.

P! I-I

e -.Ol I- 5 P! 2-2 E s-02 . 2 Q

-03 v ~ I .,I I I I I 1 2 3 4 5 6 7

PH

FIG. 3. Effect of pH on the intensities of the tyrosyl (0) and tryptophanyl (0) difference absorption peaks of Pt l-l (upper) and Pt 2-2 (lower). Experimental data are given in Fig. 2. The tyrosyl intensity was measured at 284 nm. The tryptophanyl intensity was taken as the difference between 290 and 294 nm in order to avoid including the effect of the red shift that occurs in this region.

remained constant for 3 hours. If a value of 2400 is used for the molar extinction coefficient of ionized tyrosyl residues at 295 nm (21), the change in absorption at pH 13.0 represents the dissocia- tion of 12 of the 20 phenolic groups present in both proteins.2 Since no time effects were observed and only 60% of the tyrosyl residues ionized, monkey prealbumin appears to be stable at pH values as high as 13.0. It was previously shown by several criteria that human prealbumin was stable at least to pH 12 (5, 19).

Fluorescence-Pt l-l and Pt 2-2 both show emission maxima

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100 I I I I I I 00 Pt I-1 AA Pt2-2

PH

FIG. 4. Tyrosyl titration of monkey prealbumin (0.5 mg per ml) solutions in 0.05 M ohosahate-0.1 M KC1 as measured bv the in- crease in absorbance at 295 nm. A molar extinction increase of 2400 was used to calculate the number of ionized tyrosyl residues. l , Pt l-l A, Pt 2-2. Temperature = 25”.

I I I I I 80

1

I I I , ’ \]

0 2 4 6 8 IO 12

PH

FIG. 5. The pH dependence of the fluorescence emission intensi- ties of tyrosyl (305 nm) and tryptophanyl (340 nm) chromophores. Protein concentrations were 0.025 mg per ml of 0.05 M phosphate- 0.1 M KCl. The fluorescence is plotted in arbitrary units. 0, Pt l-l; A, Pt 2-2. Temperature = 25”.

at 338 nm when excited at 280 nm. This value is similar to that found for human prealbumin (5). The emission peak did not change significantly when the pH was lowered from 7.4 to 2.4. The pH dependence of the fluorescence intensities of the tyrosyl and tryptophanyl residues, measured at 305 and 340 nm, respec- tively, is seen in Fig. 5. The tyrosyl emissions of the two forms of monkey prealbumin resemble each other, whereas trypto- phanyl emission diverges more strongly in acid. There is also an important difference in alkali since the fluorescence of Pt l-l and Pt 2-2 is quenched by 25 and 50%, respectively, between pH 8 and 12. The change in tryptophan fluorescence in both proteins is closely paralleled by a decrease in tyrosyl fluorescence. In the absence of a structural modification below pH 12, the quenching of both tyrosyl and tryptophanyl fluorescence could result from energy transfer to ionized tyrosyl residues (29, 30). The critical transfer distances (Ro) are 15.2 A for Tyr + Tyr- and 13.3 A for Trp --f Tyr-.

Polarization of Dns Fluorescence-Both preparations of con-

I OO

I I I I I 2 4 6 8 IO

1 10.10 12

PH

FIG. 6. The pH dependence of the polarization (O ,A) and fluorescence (0, A) of Dns-prealbumin (0.02 mg per ml) in 0.05 M phosphate-O.1 M KCl. The excitation and emission wave lengths were at 340 and 500 nm, respectively. Temperature = ‘,.”

jugated prealbumin contained about 2 moles of Dns per mole of protein. The emission peaks of the Dns label were at 510 and 495 nm at pH 7.4 for Pt l-l and Pt 2-2, respectively, and both shifted to 515 nm at pH 3.0. Fig. 6 shows the fluorescence intensity and polarization values obtained by titrating neutral solutions to either pH 1.8 or 12 at 25”. The polarization of neither protein changed significantly between pH 1.8 and 12. The fluorescence intensity, however, increased by about 50% between pH 7.0 and 4.0 in both proteins. The quenching of Dns fluorescence observed below pH 4 is presumably due to protonation of the amino group of Dns (31).

When a Dns-labeled human prealbumin preparation3 was studied, the pH-Dns fluorescence curve was quite different from that of monkey prealbumin. The emission peak was at 500 nm at pH 7.0 and shifted to 515 nm at pH 3.0; the intensity fell monotonically by -15% between pH 7.0 and 4.0 at 500 run. At 530 nm, however, no change in fluorescence occurred between pH 7.0 and 4.0, in agreement with the data reported previously (15) for this species.

The relaxation time of Dns-prealbumin was evaluated in order to see whether the fluorescence change was accompanied by a modification in protein conformation. A linear dependence of l/P + 36 on T/q was observed as the temperature was increased from 1045” (Fig. 7). The data for both proteins at bot’h pH values give a PO value equal to 0.27. A value of 19 f 1 ns was found for the fluorescence lifetime of Dns for both proteins at pH 7.0 which increased to 23 f 1 ns (for both proteins) at pH 3.0. In contrast to the fluorescence behavior, the relaxation time (Ph), calculated from the Perrin equation, did not change between pH 7.0 and 3.0 in either protein. There is, however, a small difference in relaxation time between the two proteins since values of 6.7 x 10B8 and 8.3 x lop8 s were found for Pt l-l and Pt 2-2, respectively. The relaxation time (~0~~‘) of a sphere of molecular weight 54,000 and 6 = 0.73 is 4.2 X low8 s by the Stokes equation. Consequently, the &PO is 1.6 and 1.9 for Pt l-l and Pt 2-2, respectively. The slightly higher value for Pt 2-2 may be due to a small amount of aggregation. A rat,io of 1.6 was previously found for human prealbumin (5).

3 The human Dns-prealbumin was prepared by Dr. R. Ferguson and contained 1.3 moles of Dns per mole of protein as determined from the radioactivity of the tritriated Dns.

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--Im +

-IL

FIG. 7. Perrin plots of the polarization of fluorescence of Dns conjugates of monkey prealbumin (0.02 mg per ml) at pH 7.0 (0 ,A) and pH 3.0 (0 ,A) in 0.05 M phosphate-O.1 M KCl. The polarization was measured as the temperature was increased from 10 to 45”.

I I I I I

280 290 300 310 320

WAVELENGTH (nm)

FIG. 8. The effect of 7.3 M guanidine HCl on the difference absorption spectrum of Pt 1-l (0.35 mg per ml) in 0.05 M phosphate- 0.1 M KCl. The spectrum at pH 8 was obtained 2 hours after adding guanidine HCI, while the one at pH 2 was measured about 10 min after the pH 8 solution was acidified with HCl.

Stability in Guanidine

Human prealbumin is exceptionally stable towards denatura- tion in concentrated urea and guanidine solutions (19). Con- centrations of urea as high as 8 M had no effect on the structure of human prealbumin. Guanidine concentrations greater than 6 M were needed to denature prealbumin at significant rates (19). It was therefore of interest to compare the stability of the mon- key proteins with that of the human. Fig. 8 shows the ultra- violet difference spectrum of Pt l-l after incubation for 2 hours in 7.3 M guanidine HCI at pH 8.0. The effect of the same condi- tions on Pt 2-2 was indistinguishable from that on Pt l-l. There is a negative difference peak at 285 nm with AeSss/ezso (pH 8)

equal to 0.053. Kinetic experiment.s were done at different concentrations of

guanidine HCl in order to compare the stability of the two

8 .8

-z .7 ii- a .6 I

I I I I I I

0 IO 20 30

MINUTES

FIG. 9. The effect of 5.6, 6.6, and 7.3 M guanidine HCl at pH 8.0 on the rate of denaturation of Pt l-l (0) and Pt 2-2 (A) (0.35 mg per ml) in 0.05 M phosphate-O.1 M KCI. The decrease in ab- sorption at 285 nm is plotted as a first order process. The total change in absorption, AA,, was obtained by acidification to pH 2.0. Temperature = 25”.

shows data for the two monkey proteins where the residual absorbance change, (1-AA,/AA,), is plotted on a semilog scale against time. The rates of denaturation of Pt l-l and Pt 2-2 increase strongly between 5.6 and 7.3 M guanidine HCI. The kinetic curves are complex since they do not follow first order kinetics, as seen in Fig. 9. At all three guanidine HCl concen- trations, however, Pt 2-2 had a slightly slower denaturation rate than Pt 1-1. It was noted during these experiments that the magnitude of the difference spectra change (AE~&E& of both proteins doubled upon acidification of the concentrated guanidine HCl solution from pH 8.0 to 2.0. Fig. 8 shows the effect of pH on the difference spectra of Pt l-l in 7.3 M guanidine HCI.

The effect of acidification on the difference absorption at 285 nm of solutions of monkey prealbumin in 7.3 M guanidine HCl was evaluated further. The absorbance change of Pt l-l reached a plateau after 5 min at pH 8.0 (Fig. 10). When the pH was adjusted to 4.0, the absorbance changed further and required 45 min to approach a limit (Fig. 10). Similar results were obtained with Pt 2-2. The monkey proteins, therefore, display a two-step denaturation process in 7.3 M guanidine HCl, i.e. one at pH 8.0 and a second one at lower pH values. This behavior is strikingly different from that observed with human prealbumin, where the absorbance change reached its final value in several minutes in 7.3 M guanidine HCl at pH 8.0 and almost no further change was observed upon acidification to pH 2.0 (Fig. 10).

Binding of Thyroxine and Retinal-binding Protein

Equilibrium dialysis data for thyroxine binding to Pt l-l and Pt 2-2, plotted according to the Scatchard equation, are shown in Fig. 11. There appears to be no difference in thyroxine binding between the two proteins. The line in Fig. 11, calcu- lated by the method of least squares, represents an association constant of 0.85 X lo8 Me' with n = 0.85 for the two proteins. Estimates of the association constant for the interaction of thyroxine with human prealbumin have varied from 0.16 to 3.6 x lo8 M? (8-12). A value of 1.3 X lo* M? was obtained for human prealbumin4 by equilibrium dialysis performed under similar experimental conditions as with the monkey proteins.

It has been reported that human prealbumin binds 1 mole of RBP (13-15). We have recently demonstrated that there are four RBP binding sites of the same affinity (16). Fig. 12 shows

monkey proteins. The change in absorbance at 285 nm was followed in 5.6, 6.6, and 7.3 M guanidine HCI (pH 8.0). Fig. 9

4 R. A. Pages, J. Robbins, and H. Edelhoch, manuscript in preparation.

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IO I , I 1 / I I .0 7:3 M Gu HCI

.Pl ' I I I I I 0 IO 20 30 40

MINUTES

FIG. 10. The effect of pH on the extent of denaturation in 7.3 M guanidine HCl at pH 8.0 of Pt l-l (0) and human prealbumin (0) (0.35 mg per ml) in 0.05 M phosphate-O.1 M KCl. After 15 min the pH of the Pt l-l solution was brought to 4.0 with HCl. Acidification of human prealbumin to pH 2 had no further effect on its difference absorption. Other details are the same as in Fig. 9.

r I I I I 06 Pt I-I ar Pt 2-2

a

A

?I

0

A

OL I 0 .2 %4 6 .8 I .o

v FIG. 11. Scatchard plot of thyroxine binding data obtained by

equilibrium dialysis at pH 7.4; Pt l-l (0, l ) and Pt 2-2 @,A) (0.79 PM) in 0.05 M phosphate-O.1 M KCl-1 mM EDTA. Tempera- ture = 25”. See text for significance of open and jilled symbols.

the effect of monkey prealbumin on the polarization of fluores- cence of retinol bound to RBP. The data have been analyzed by a curve fitting program. The open and closed symbols in Fig. 12 represent data for Pt l-l and Pt 2-2, respectively; the lines are the theoret,ical binding curves for values of K = 1.4, 1.0, and 0.93 x lo6 M-~ (n = 4) at Ohe three RBP concentrations. No difference in the binding properties of the two monkey proteins is apparent over a 5-fold range in RBP concentration (1.1 to 5.1 PM). The K values are essentially the same as found with human prealbumin (16).

DISCUSSION

The amino acid compositions of the two monkey proteins indicate an interchange in 1 residue, i.e. valine for isoleucine.2 Inasmuch as the total number of amino acids is the same in the

a

4

l CI

.+.+I

I

f

A

r

3 %O 0.2 0.Y 0.6 0.6 1.0 1.2

FIG. 12. The binding of retinol-binding protein by prealbumin at pH 7.4. The dependence of polarization of retinol fluorescence of human RBP on prealbumin to RBP molar ratios; Pt 1-1, open symbols; Pt 2-2, closed symbols. Solvent was 0.05 M phosphate-O.1 M KCl; RBP concentration: 0 , 0, 5.1; A,& 2.3; 0, n , 1.1 pM.

The lines are theoretical best fit curves with n = 4 and K = 1.4 (0, l ), 1.0 @,A), and 0.9 X lo6 M-I (Ll,@. Polarization was measured at 28”.

two proteins, this could represent a one-point mutation. Since; however, Pt l-l and Pt 2-2 differ in electrophoretic mobility, there is possibly also a difference in amide groups. The available data are not yet sufficient to specify the exact number of sub- stitutions. The amino acid composition of human prealbumin reveals more differences between it and monkey prealbumin than exist between the two monkey proteins.

Exchanges between amino acids with nonpolar side chains, which are likely to be interior residues, can affect the interactions within the protein, If these occur in conformationally sensitive regions of the structure they can lead to modified physical and chemical properties of the protein. The two different types of binding sites are apparently unaltered by the exchange of residues since the affinities of Pt l-l and Pt 2-2 for thyroxine and RBP are the same. The 4 RBP molecules evidently bind to the 4 subunits (16). Since thyroxine and RBP bind to different sites of prealbumin the single thyroxine-binding site is probably in the channel created by the 4 subunits of prealbumin (3). Since none of the sites is altered, it is likely that the organization of the polypeptide backbone responsible for these five binding sites is not significantly different in the two monkey proteins. In fact, the CD spectra in the far ultraviolet show no differences in the secondary structure of the entire polypeptide chain.

If the secondary structure is unchanged, the effects of amino acid substitutions must be looked for in the tertiary structure. The latter has been evaluated from the behavior of the two intrinsic chromophores, the tyrosyl and tryptophanyl residues, and an extrinsic chromophore, Dns. The absorption, and especially the fluorescence properties, of all three groups are sensitive to their environments and can be used as structural probes (23). In human prealbumin the far ultraviolet circular dichroic spectrum does not change between pH 2 and 12, but there are significant blue shifts in the absorption properties of the aromatic chromophores between pH 3.5 and 2.5 (5). The two monkey proteins show very similar blue shifts in the same pH region. The intensities of the difference absorption peaks of

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the tyrosyl and tryptophanyl residues are, however, slightly different in Pt l-l and Pt 2-2. The fluorescence behavior of these two chromophores in both acid and alkaline pH is also different in Pt l-l and Pt 2-2. Thus the interactions of aromatic groups with their environments are modified in the two forms of monkey prealbumin. It is unlikely, however, that all of the 5 tyrosyl and 3 tryptophanyl residues per subunit are affected since the difference in behavior between the two proteins is relatively small.

The pH effects observed between 3.5 and 2.5 by absorption in the native protein are not encountered in the fluorescence behavior of Dns in the conjugated protein. There is, however, a transition between pH 7.0 and 3.0 causing an increase in Dns fluorescence intensity and lifetime. This enhancement in quantum yield of Dns represents only a local environmental effect since the relaxation time of prealbumin does not vary between pH 7.0 and 3.0. Monkey prealbumin molecules re- semble human prealbumin in their high values for &PO, indi- cating a lack of rotational freedom between subunits. Similarly, the resistance of monkey and human prealbumin to dissociation by concentrated guanidine HCI solutions indicates that the intersubunit interactions are unusually strong. Other proteins composed of subunits show &PO values less than one (32, 33).

The pH dependence of the acid transition and the effect of guanidine HCl on the rate of denaturation at pH 8 are similar for the two monkey proteins. There is, however, a significant difference between monkey and human prealbumin in the extent of denaturation. The difference spectra in acid and in 7.3 M

guanidine HCl at pH 8 are about half as intense in monkey as in human prealbumin. It is not surprising that the acid transition does not normalize the accessibility of all of the tyrosyl and tryptophanyl residues in monkey prealbumin since this transition does not change the rotational behavior of the protein. On the other hand, it is very unusual to find that about half of the aromatic residues remain inaccessible after denaturation in 7.3 M

guanidine HCI. These residues become exposed to the solvent in a second transition produced by acidification below pH 4. There are, therefore, at least two distinct molecular transitions and probably three since the difference spectra are not the same at pH 2 as in 7.3 M guanidine HCI at pH 8. Acidification of the 7.3 M guanidine HCl solution probably destroys all of the residual interactions and produces a molecule which has a random coil structure since there are no disulfide bonds in prealbumin.

There are only a few differences in the ammo acid composition of the genetic variants of monkey prealbumin and human pre- albumin. These have essentially no effect on the folding of the polypeptide chain, on the structures responsible for thyroxine and retinol-binding protein binding, or on the subunit interac- tions. Although minor differences between the two monkey proteins were seen in their denaturation by acid or guanidine HCl, the only important difference observed was between monkey and human prealbumin. The two monkey proteins still retain structured elements in 7.3 M guanidine HCl since they undergo further denaturation blue shifts when exposed to acid. This two-stage molecular unfolding was not observed with human prealbumin.

Acknowledgments-We thank Dr. Raymond F. Chen for the use of his fluorescence decav time annaratus: Drs. Robert A.

Pages and Robert Goebel for their help with the equilibrium dialysis, and Dr. H. A. Saroff for the programs used to evaluate retinol-binding protein binding to prealbumin. We are also indebted to Drs. Dew. S. Goodman and Robert Ferguson for gifts of human retinal-binding protein and human Dns-prealbu- min.

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Pieter P. van Jaarsveld, William T. Branch, Harold Edelhoch and Jacob RobbinsPROTEIN

PROPERTIES AND BINDING OF THYROXINE AND RETINOL-BINDING Polymorphism of Rhesus Monkey Serum Prealbumin: MOLECULAR

1973, 248:4706-4712.J. Biol. Chem. 

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