Location of tryptophyl and tyrosyl residues in human chorionic gonadotropin

BIOCHIMICAET BIOPHYSICAACTA

523

BBA R e p o r t BBA 31125 L o c a t i o n o f t r y p t o p h y l and t y r o s y l residues in h u m a n c h o r i o n i c g o n a d o t r o p i n

K. FRED MORI Food and Drug Research Laboratories, Department of National Health and Welfare, Ottawa, Ontario KIA OL2 (Canada)

(Received January 10th, 1972)

SUMMARY

A solvent perturbation study of human chorionic gonadotropin showed that in the native form about four of the seven tyrosyl residues and about 20% of the single tryptophyl residue were exposed to perturbants (2H20 , ethylene glycol, sucrose) and that two more tyrosyls became available to perturbation with ethylene glycol in the presence of 8 M urea. Complete exposure of these two chromophoric residues to this perturbant was achieved in the disulfide-cleaved protein. Human chorionic gonadotropin contains one tryptophyl and seven tyrosyl residues per mole (mol. wt = 37 700) 1, but only for of the seven tyrosyls were found to be reversibly titratable under normal conditions 2. Further studies were conducted to characterize the location of not only tyrosyl but also tryptophyl residues in this hormone by the use of the solvent perturbation technic 3. An apparently homogeneous human chorionic gonadotropin preparation was obtained from a commercial product (3300 I.U./mg, Organon, Inc.) as described previously 1and was assayed to contain a biological activity of 15 800 I.U./mg by the seminal vesicle wt method 4. Difference spectra of human chorionic gonadotropin in a few perturbants were read in a Cary Model 15 recording spectrophotometer as described by Herskovits and Laskowski s . The perturbants used were 2H20, ethylene glycol and sucrose. Biological activities of human chorionic gonadotropin solutions in 0.1 M KCI, pH 7.5, containing each perturbant were essentially the same as that of an human chorionic gonadotropin solution in 0.1 M KC1, pH 7.5. Protein concentrations were spectrophotometricaUy determined using ~lcm ~.1% = 5.47 at 276 nm 2, Difference spectral measurements of disulfidecleaved human chorionic gonadotropin were made in 8 M urea in the presence of #-mercaptoethanol (3/al/mg protein). In all cases the original readings from three independent measurements were used to compute Ae M values, the means of which are Biochim. Biophys. Acta, 257 (1972) 523-526

I,J

I

to

I%

TABLE I

687

550

-106 157 87

X (nm) 290--292

Experimental

1037

793

-301 389 218

k {nm) 285-288

649

649

-323 455 246

k (nm) 290--292

1017

1017

-596 817 441

k (nmJ 285-288

Model compound*

*Calculated according to Eqn 3, using the model compound data of Herskovits and Sorensen 6. **Based on the ratio of the experimental Ae M values of the protein to the calculated model values.

Mercaptoethanol-containing 8 M urea, 0.1 M CI-, pH 7.5 20% ethylene glycol

8 M urea, 0.1 M CI-, pH 7.5 20% ethylene glycol

0.1 M KCI in 0.01 M phosphate buffer, pH 7.5 90% deuterium oxide 20% ethylene glycol 20% sucrose

Solven t and perturban t

Molar absorbance differences { A eMJ

DIFFERENCE SPECTRA PARAMETERS OF HUMAN CHORIONIC GONADOTROPIN

1.06

0.85

0.33 0.35 0.35

Trp

1.02

0.78

0.51 0.48 0.50

Tyr

Fraction o f residues exposed * *

0

>

tO

BBA REPORT

525

presented in Table I. Approximate values for the degree o f the t r y p t o p h y l and tyrosyl exposure were estimated b y solving the following two equations 6' 7. Ae290---292 nm (protein) =aAe290--292 nm (Trp) + bAe290--292 nm (Tyr) Ae285--288 nm (protein) = a A e 2 s s - 2 s 8 nm ( T r p ) + b A e 2 s s - 2 a s nm (Tyr)

(1) (2)

where a and b represent the apparent number of exposed t r y p t o p h y l (Trp) and tyrosyl (Tyr) residues in human chorionic gonadotropin. Values o f the parameters involved in the solvent perturbation study o f human chorionic gonadotropin are summarized in Table I. The estimates o f a and b obtained with 20% ethylene glycol were refmed to produce the best fit o f the observed difference spectra with the calculated ones by the use o f Ae M, X = 1 X Ae M, X (Trp) + 7 X Ae M, ?t (Tyr) I

I

I

I

S-S cleaved J l ~ i n 8 M urea

1 0 0 0 --

(3)

i -

gOC

60C

40C

I

20C

//

~k

El

BOC

I

I

I

-

/ I!8

M urea

60C

40C

~

20C

I

I

270

280

I 290 ;~(nm)

_

I 300

310

Fig. 1. Solvent perturbation difference spectra of native (pH 7.5, 0.1 M KCI), 8 M urea-denatured (pH 7.5, 0.1 M KCI), and disuLfide-cleaved (in 8 M urea, pH 7.5, 0.1 M KCI) human chorionic gonadotropin produced by 20% ethylene glycol. Solid lines represent the experimental data, whereas dashed lines show the theoretical spectra calculated from Eqn 3 with the following values of a and/7: a = 0.2, b -- 3.6 for native; a = 0.8, b = 6.2 for denatured; and a = 1, b = 7 for disulfide-cleaved human chorionic gonadotropin. Protein concentrations varied from 3.3.10 -`5 to 5.3.10 -'s M. Biochim. Biophys. Acts, 257 (1972) 523-526

526

BBAREPORT

as described by Herskovits and Sorensen 6. The results are shown in Fig. 1. The three perturbants, of different molecular diameter, yielded essentially the same values for the degree of tryptophyl and tyrosyl exposure, suggesting that a very small fraction of the chromophores are located in crevices or intrasurfaces of the protein tertiary structure, if any, formed by the peptide fold. In native human chorionic gonadotropin about 50%, i.e. four of the seven tyrosyls, were exposed to the solvent. This value corresponds very well with that estimated from the titration study 2 . On the other hand, the single tryptophyl residue was exposed only to an extent of about 20%. Urea apparently unfolded the human chorionic gonadotropin molecule to render the two chromophoric residues more accessible to the perturbant molecules. However, the tryptophyl and one of the seven tyrosyl residues were still incompletely exposed under this condition. Complete agreement between the observed and calculated complete spectra was obtained only in the disulfide-cleaved protein. Since human chorionic gonadotropin is characterized by a high content of disulfide bonds, the tryptophyl and this particular tyrosyl residue are likely to be adjacent to one of the disulfide bridges of the hormone molecule. Since human chorionic gonadotropin in the presence of 8 M urea or its disulfide-cleaved product was found to be totally biologically inactive as examined by the ovarian hyperemia assay method s, the one tryptophyl and those three tyrosyls which became exposed upon unfolding of the molecule, are presumably buried inside an as yet unknown tertiary structure that is required for the manifestation of biological activity of this hormone. Mrs V.G. Hum provided invaluable technical assistance.

REFERENCES 1 2 3 4 5 6 7 8

K.F. Mori, Endocrinology, 86 (1970) 97. K.F. Moil and T.R. HoUands,J. Biol. Chem., 246 (1971) 7223. T.T. Herskovits,Methods Enzymol., 11 (1967) 748. H. van Hell, R. Matthijsen and G.A. Overbeek, Acta Endocrinol., 47 (1964) 409. T.T. Herskovits and M. Laskowski, Jr.,J. Biol. Chem. 237 (1962) 2481. T.T. Herskovits and M. Sorensen,Biochemistry, 7 (1968) 2523. T.T. Herskovits and M. Sorensen,Biochemistry, 7 (1968) 2533. A. Albert and J. Berkson, J. Clin. Endocrinol. Metab., 11 (1951) 805.

Biochim. Biophys. Acta, 257 (I~,72) 523-526