- Email: [email protected]
Circular dichroism studies on ovine follicle stimulating hormone
142
SHORT COMMUNICATIONS
the 95 ~o confidence limits of the d e t e r m i n a t i o n of the g-potentials b y electrophoretic m e a s u r e m e n t s , which accounts for the fact t h a t it could not be observed. Since the electrical charge is p r o p o r t i o n a l to ~-, the charges of asl-casein B a n d C also differ by 8.50/o . The m e a n charge of the monomers, calculated from their electrophoretic mobility, is --19. 5 units, so t h a t the calculated charge difference is 1. 7 units. This difference is higher t h a n might be expected on the basis of the amino acid composition of the asl-casein variants, b u t in view of the a p p r o x i m a t i o n s t h a t had to be made, the agreement is satisfactory. We therefore conclude t h a t the differences in association behaviour observed with the genetic variants, B, C, a n d D of asl-casein m a y be ascribed to the electrical charge difference introduced b y the a m i n o acid replacements in the polypeptide chain. Netherlands Institute for Dairy Research, Ede (the Netherlands)
D. G. SCHMIDT
A. J. PAYENSAND D. G. SCHMIDT,Biochim. Biophys. Acta, lO9 (1965) 214. A. J. PAYENSAND D. G, SCHMIDT,Arch. Biochem. Biophys., 115 (1966) 136. 3 H. A. McKENzlE, Adv. Prot. Chem., 22 (1967) 56. 4 D • G. SCHMIDTAND ]3. W. VAN MARKWIJK,Biochim. Biophys. Acta, 154 (1968) 61o. 5 H . E. S~,VAISGOODAND S. N. TIMASHEFF,Arch. Biochem. Biophys., 125 (1968) 334. 6 D . G. SCHMIDT, Biochim. Biophys. Acta, 207 (197o) 13o. 7 D . G. SCHMIDTAND T. A. J. PAYENS, Neth. Milk Dairy J., 18 (1964) lO8. 8 P. J. DE KONINGAND P. J, VAN ROOYEN, Nature, 213 (1967) lO28. 9 P . J. D~ KONING, Thesis, Amsterdam, 1967. 1o D. G. SCHMIDTAND T. A. J. PAYENS, Biochim. Biophys. Acta, 78 (1963) 492. l i D . G. SCHMIDT,Thesis, Utrecht, 1969. 12 A. L. LOEB, J. TH. G. OVERBEEKAND P. H. WIERSEMA,The Electrical Double Layer around a Spherical Colloidal Particle, The MIT Press, Cambridge, Mass., 1961, p. 75. 13 P. H. WIERSEMA, A. L. LOEB AND J. TH. G. OVERBEEK,J. Colloid Sci., 22 (1966) 78. i T. 2 T.
Received J u n e 30th, 1970 Biochim. Biophys. Acta, 221 (197o) 14o-142
BBA 33234
Circular dichroism studies on ovine follicle stimulating hormone JIRGENSONS1 had previously reported, from optical r o t a r y dispersion measurem e n t s at a limited n u m b e r of wavelengths, t h a t ovine follicle s t i m u l a t i n g hormone (FSH) appeared to c o n t a i n a low c o n t e n t of secondary structure, p r e s u m a b l y a-helical in nature. I n this study, circular dichroism (CD) measurements, with their greater intrinsic resolving power, were u n d e r t a k e n with a highly purified F S H preparation to provide a more u n i q u i v o c a l estimate of the n a t u r e a n d e x t e n t of this secondary structure, and to provide some information on the t e r t i a r y structure in the i m m e d i a t e Abbreviations: FSH, follicle stimulating hormone; CD, circular dichroism. Following the CD analysis, the solutions were dialyzed to remove urea and alkali, respectively, and were bioassayed. Both showed a marked reduction of biological activity. However, a solution of FSH in 8 M urea kept in the dark for an equivalent period of time before assay did not show appreciable loss of potency. ]¢ioch;m. Biophys. Acta, 221 (197o) 142-145
SHORT COMMUNICATIONS
I43
vicinity of tile aromatic residues 2. In addition, the extent and reversibility of denaturation of the protein by alkaline pH, and 8 M urea have been investigated. A Cary Model 6o spectropolarimeter equipped with a Model 6oo2 circular dichroism attachment was used for obtaining the CD spectra. The instrument was calibrated with (+)-camphorsulfonic acid (Eastman Organic Chemicals) as recommended by the manufacturer. All spectra were taken at 2 7 ° , in quartz cells with pathlengths ranging from i.oo mm to 2.oo cm. The FSH was prepared by a modification 3 of the procedure previously described from this laboratory 4, and was identical with respect to homogenity and chemical composition to the FSH previously described. It had a biological activity of about 45 times the National Institutes of Health standard, NIH-FSH-SI. CD spectra were taken of the native protein in o.z M NH4HCO 3 buffer, pH 8.3, at concentrations ranging from o.z to I.o mg/ml. Protein concentrations were deO I °/ termined spectrophotometrically. The E~go"~m of FSH was determined to be o.73o for ioo% protein (assuming z 5 % moisture in the lyophilized material). The solution containing 8 M urea in o.I M NH4HCO a buffer, pH 8.3, was allowed to stand at 21 ° for 3 h prior to taking its CD spectrum. The spectrum at pH I2.9 was obtained by titrating the native protein solution to this pH with zo M KOH after completion of the "native" spectrum at pH 8.3. After completing the spectra of the urea- and alkalitreated FSH, both solutions were thoroughly dialyzed against the o.i M NH4HCO 3 buffer, pH 8.3. The protein concentrations were redetermined and the CD spectra retaken. All spectra were taken from 3~5 nm to as close to 2oo nm as possible. Mean residue molecular ellipticities, [0] M~w, were calculated using z I5 for the mean residue weight and I 7 % for the carbohydrate content of ovine FSH. The contribution to the CD measurements due to the presence of carbohydrate was not assessed. The a-helix contents of the samples were estimated from the ellipticities at 2o 9 nm and 22I nm as described elsewhere a. The CD spectra of the native, alkali-, and urea-treated proteins, in the region
~i
/"
/
~l~
,,
o~, ~
i*i
100k 71o
~20
~30
W A V E L E N G I H (.m)
20
250 W A V E L E N G I H into
Fig. i. C D s p e c t r a o f n a t i v e ( - - ) , a n d u r e a - t r e a t e d ( - - . - - ) F S H in o . i M N H 4 H C O a buffer, p H 8.3; a n d n a t i v e F S H a t p H 12.9 ( - - - - ) . S i g n a l - t o - n o i s e r a t i o w a s i o : i a t 23 ° n m , 18:1 a t 2 2 o n m a n d i i : i a t 2IO n m . Fig. 2. C D s p e c t r a of n a t i v e ( - - ) , a n d u r e a - t r e a t e d ( - - - - - ) F S H in o . i M N H 4 H C O a buffer, p H 8.3; a n d n a t i v e F S H a t p H 12.9 ( - - - - ) . S i g n a l - t o - n o i s e r a t i o w a s 2.6:1 a t 283 n m , 4:1 a t 274 n m a n d 2.7:1 a t 267 n m .
Biochi~n. Biophys. Acta, 2 2 I (I97o) z 4 2 - I 4 5
144
SHORT COMMUNICATIONS
of peptide bond absorption, are shown in Fig. I. The native protein shows two of three optically active bands observed for a-helical polypeptides2, ~. Due to excessive absorption below 205 nm we have been unable to demonstrate the third positive a-helix band near 191 nm. The a-helix content of native ovine FSH estimated from this spectrum is approx. 3o% (Table I). Both the alkali- and urea-treated FSH show a diminished negative ellipticity in the region from 215 to 25o nm. Excessive absorption in both cases made accurate measurements below 21o nm impossible. However, since a completely random coil would be expected to show positive ellipticities around 220 nm2, 6, we can safely conclude that not all of the secondary structure has been destroyed by these denaturants. The helix contents of these partially denatured proteins, estimated from the ellipticities at 22o nm, are presented in Table I. Dialysis of the urea-treated sample resulted in a complete recovery of the CD spectrum from 250 to 2o5 nm to that of the native protein, indicating that the alteration in secondary structure brought about by urea is completely reversible*. This was not TABLE I ELLIPTICITIES AND ESTIMATED HELIX CONTENTS OF NATIVE AND TREATED OVINE F S H E x t r e m e s c a r c i t y of h i g h l y p u r i f i e d m a t e r i a l a l l o w e d o n l y for s i n g l e d e t e r m i n a t i o n s a t t h i s t i m e . T h e v a l u e s o f e l l i p t i c i t i e s a r e a r b i t r a r i l y a s s u m e d t o b e ± 5 ~o o f t h e n o m i n a l v a l u e . T h e v a l u e s a r e c o m p u t e d o n t h e a s s u m p t i o n t h a t t h e m o i s t u r e c o n t e n t o f t h e p r o t e i n is 15 °/o.
(O) MnW (degrees. cm 2. decimole -1) °/o Excess* right hand a-helix*
Sample
210
N a t i v e , p H 8. 3 Urea treated A l k a l i t r e a t e d , p H 12.9 Dialyzed from 8 M urea D i a l y z e d f r o m p H 12.9
~m
220
12 2oo ~ 6oo --i i 500 ~ 575 8 o o o ± 400
* Expressed to the nearest
9 4 5 9 5
n•
9oo ooo ioo 200 800
210
± J= ~ ~ ±
5oo 2oo 250 45 ° 300
3° --25 20
~'lm
220
nm
3° 2o 20 3° 20
5/o.°/
true of the alkali-denatured sample which showed only a partial return of its spectrum to that of the native protein. Ellipticities and estimated helical contents of these dialyzed samples are given in Table I. The CD spectrum of the protein in the region of side-chain absorption is shown in Fig. 2. At pH 8. 3 the spectrum of the native protein shows a weak negative shoulder at 293-294 nm, two overlapping negative bands with apparent peaks at 283 and 273-275 nm, and two additional negative peaks at 267 and 262 nm : the strong negative beginning at about 258 nm is mostly due to the a-helix band at 22o nm. The spectrum of the urea-treated protein is shifted towards less negative ellipticity values from that of the native but shows essentially the same pattern of optically active bands. Upon removal of the urea by dialysis, this portion of the spectrum does not return to that of the native protein, in contrast to the spectrum in the region of peptide-bond absorption. The spectrum at p H I2. 9 shows a complete loss of the negative dichroism exhibited by the native protein from 325 to 275 nm. This is replaced by a fairly strong positive band centered around 25o nm. On dialysis of this material back to pH 8.3 Biochim. Biophys. Aeta, 221 (197 o) i 4 2 - I 4 5
145
SHORT COMMUNICATIONS
this positive peak disappears but with very little reformation of the negative dichroism bands between 260 and 325 nm. As discussed by BEYCHOKz, diehroism in the region from 26o to 325 nm is mainly due to the side chains of the aromatic amino acids, and is very sensitive to the tertiary structure in the immediate vicinity of these residues. The CD spectra of native FSH shows a number of optically active bands in this region. The two negative bands at 267 and 262 nm m a y be tentatively assigned to phenylalanine by comparison with the CD spectra of N-benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine in lOO% ethylene glycoF. The two negative bands at 273-275 and 283 nm, and the negative shoulder at 295 nm m a y be due either to tyrosine, tryptophan or possibly both. The positive peak at 25 ° n m seen at pH 12. 9 is ahnost certainly due to ionized tyrosine residues2, 8. These studies indicate that between one-fourth and one-third of the amino acid residues in FSH are in an a-helical conformation. This secondary structure m a y be partially denatured by treatment with either strong alkali or 8 M urea. While strong alkali appears to bring about an irreversible loss of secondary structure, the loss of a-helix caused by urea treatment is completely reversible. Both treatments produce alterations in the tertiary structure of the molecule as shown by the altered environments of the aromatic residues. However, it should be noted that the effect of urea on the tertiary structure is both less extensive and more nearly reversible than that of alkali. We thank Professor C. H. Li for suggesting this study and for his advice. The work was supported in part by the U.S. Public Health Service Grant A-6o97 from the National Institute of Arthritis and Metabolic Diseases and the Geffen Foundation. One of us (H.P.) is a Career Development Awardee, Institute of General Medical Science, U.S. Public Health Service.
Hormone Research Laboratory, University of California, San Francisco, Cahf. (U.S.A.) I 2 3 4 5 6 7 N
MARGARETA EKBLAD
THOMAS A. BEWLEY HAROLD PAPKOFF
B. JIRGEXSONS, Arch. Biochem. Biophys., 91 (196o) 123. S. BEYCHOK, Science, 154 (1966) 1288. M. EKBLAD, H. PAPKOFF AND C. H. LI, to be published. H. PAPKOFF, D. GOSPODAROWICZ AND C. H. LI, Arch. Biochem. Biophys., 12o (1967) 434. T. A. BEWLEY AND C. H. LI, Arch. Biochem. Biophys., 138 (197 o) 338. G. HOLZWORTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. I. WEINRYB AND R. F. STEINER, Arch. Biochem. Biophys., 131 (1969) 263. N. S. SIMMONS AND A. N. GLAZER, J. Atn. Chem. Soc., 89 (1967) 5o4 o.
Received May I9th, 197o Biochim. Biophys. Acta, 22i (197 o) 142-145
SHORT COMMUNICATIONS
the 95 ~o confidence limits of the d e t e r m i n a t i o n of the g-potentials b y electrophoretic m e a s u r e m e n t s , which accounts for the fact t h a t it could not be observed. Since the electrical charge is p r o p o r t i o n a l to ~-, the charges of asl-casein B a n d C also differ by 8.50/o . The m e a n charge of the monomers, calculated from their electrophoretic mobility, is --19. 5 units, so t h a t the calculated charge difference is 1. 7 units. This difference is higher t h a n might be expected on the basis of the amino acid composition of the asl-casein variants, b u t in view of the a p p r o x i m a t i o n s t h a t had to be made, the agreement is satisfactory. We therefore conclude t h a t the differences in association behaviour observed with the genetic variants, B, C, a n d D of asl-casein m a y be ascribed to the electrical charge difference introduced b y the a m i n o acid replacements in the polypeptide chain. Netherlands Institute for Dairy Research, Ede (the Netherlands)
D. G. SCHMIDT
A. J. PAYENSAND D. G. SCHMIDT,Biochim. Biophys. Acta, lO9 (1965) 214. A. J. PAYENSAND D. G, SCHMIDT,Arch. Biochem. Biophys., 115 (1966) 136. 3 H. A. McKENzlE, Adv. Prot. Chem., 22 (1967) 56. 4 D • G. SCHMIDTAND ]3. W. VAN MARKWIJK,Biochim. Biophys. Acta, 154 (1968) 61o. 5 H . E. S~,VAISGOODAND S. N. TIMASHEFF,Arch. Biochem. Biophys., 125 (1968) 334. 6 D . G. SCHMIDT, Biochim. Biophys. Acta, 207 (197o) 13o. 7 D . G. SCHMIDTAND T. A. J. PAYENS, Neth. Milk Dairy J., 18 (1964) lO8. 8 P. J. DE KONINGAND P. J, VAN ROOYEN, Nature, 213 (1967) lO28. 9 P . J. D~ KONING, Thesis, Amsterdam, 1967. 1o D. G. SCHMIDTAND T. A. J. PAYENS, Biochim. Biophys. Acta, 78 (1963) 492. l i D . G. SCHMIDT,Thesis, Utrecht, 1969. 12 A. L. LOEB, J. TH. G. OVERBEEKAND P. H. WIERSEMA,The Electrical Double Layer around a Spherical Colloidal Particle, The MIT Press, Cambridge, Mass., 1961, p. 75. 13 P. H. WIERSEMA, A. L. LOEB AND J. TH. G. OVERBEEK,J. Colloid Sci., 22 (1966) 78. i T. 2 T.
Received J u n e 30th, 1970 Biochim. Biophys. Acta, 221 (197o) 14o-142
BBA 33234
Circular dichroism studies on ovine follicle stimulating hormone JIRGENSONS1 had previously reported, from optical r o t a r y dispersion measurem e n t s at a limited n u m b e r of wavelengths, t h a t ovine follicle s t i m u l a t i n g hormone (FSH) appeared to c o n t a i n a low c o n t e n t of secondary structure, p r e s u m a b l y a-helical in nature. I n this study, circular dichroism (CD) measurements, with their greater intrinsic resolving power, were u n d e r t a k e n with a highly purified F S H preparation to provide a more u n i q u i v o c a l estimate of the n a t u r e a n d e x t e n t of this secondary structure, and to provide some information on the t e r t i a r y structure in the i m m e d i a t e Abbreviations: FSH, follicle stimulating hormone; CD, circular dichroism. Following the CD analysis, the solutions were dialyzed to remove urea and alkali, respectively, and were bioassayed. Both showed a marked reduction of biological activity. However, a solution of FSH in 8 M urea kept in the dark for an equivalent period of time before assay did not show appreciable loss of potency. ]¢ioch;m. Biophys. Acta, 221 (197o) 142-145
SHORT COMMUNICATIONS
I43
vicinity of tile aromatic residues 2. In addition, the extent and reversibility of denaturation of the protein by alkaline pH, and 8 M urea have been investigated. A Cary Model 6o spectropolarimeter equipped with a Model 6oo2 circular dichroism attachment was used for obtaining the CD spectra. The instrument was calibrated with (+)-camphorsulfonic acid (Eastman Organic Chemicals) as recommended by the manufacturer. All spectra were taken at 2 7 ° , in quartz cells with pathlengths ranging from i.oo mm to 2.oo cm. The FSH was prepared by a modification 3 of the procedure previously described from this laboratory 4, and was identical with respect to homogenity and chemical composition to the FSH previously described. It had a biological activity of about 45 times the National Institutes of Health standard, NIH-FSH-SI. CD spectra were taken of the native protein in o.z M NH4HCO 3 buffer, pH 8.3, at concentrations ranging from o.z to I.o mg/ml. Protein concentrations were deO I °/ termined spectrophotometrically. The E~go"~m of FSH was determined to be o.73o for ioo% protein (assuming z 5 % moisture in the lyophilized material). The solution containing 8 M urea in o.I M NH4HCO a buffer, pH 8.3, was allowed to stand at 21 ° for 3 h prior to taking its CD spectrum. The spectrum at pH I2.9 was obtained by titrating the native protein solution to this pH with zo M KOH after completion of the "native" spectrum at pH 8.3. After completing the spectra of the urea- and alkalitreated FSH, both solutions were thoroughly dialyzed against the o.i M NH4HCO 3 buffer, pH 8.3. The protein concentrations were redetermined and the CD spectra retaken. All spectra were taken from 3~5 nm to as close to 2oo nm as possible. Mean residue molecular ellipticities, [0] M~w, were calculated using z I5 for the mean residue weight and I 7 % for the carbohydrate content of ovine FSH. The contribution to the CD measurements due to the presence of carbohydrate was not assessed. The a-helix contents of the samples were estimated from the ellipticities at 2o 9 nm and 22I nm as described elsewhere a. The CD spectra of the native, alkali-, and urea-treated proteins, in the region
~i
/"
/
~l~
,,
o~, ~
i*i
100k 71o
~20
~30
W A V E L E N G I H (.m)
20
250 W A V E L E N G I H into
Fig. i. C D s p e c t r a o f n a t i v e ( - - ) , a n d u r e a - t r e a t e d ( - - . - - ) F S H in o . i M N H 4 H C O a buffer, p H 8.3; a n d n a t i v e F S H a t p H 12.9 ( - - - - ) . S i g n a l - t o - n o i s e r a t i o w a s i o : i a t 23 ° n m , 18:1 a t 2 2 o n m a n d i i : i a t 2IO n m . Fig. 2. C D s p e c t r a of n a t i v e ( - - ) , a n d u r e a - t r e a t e d ( - - - - - ) F S H in o . i M N H 4 H C O a buffer, p H 8.3; a n d n a t i v e F S H a t p H 12.9 ( - - - - ) . S i g n a l - t o - n o i s e r a t i o w a s 2.6:1 a t 283 n m , 4:1 a t 274 n m a n d 2.7:1 a t 267 n m .
Biochi~n. Biophys. Acta, 2 2 I (I97o) z 4 2 - I 4 5
144
SHORT COMMUNICATIONS
of peptide bond absorption, are shown in Fig. I. The native protein shows two of three optically active bands observed for a-helical polypeptides2, ~. Due to excessive absorption below 205 nm we have been unable to demonstrate the third positive a-helix band near 191 nm. The a-helix content of native ovine FSH estimated from this spectrum is approx. 3o% (Table I). Both the alkali- and urea-treated FSH show a diminished negative ellipticity in the region from 215 to 25o nm. Excessive absorption in both cases made accurate measurements below 21o nm impossible. However, since a completely random coil would be expected to show positive ellipticities around 220 nm2, 6, we can safely conclude that not all of the secondary structure has been destroyed by these denaturants. The helix contents of these partially denatured proteins, estimated from the ellipticities at 22o nm, are presented in Table I. Dialysis of the urea-treated sample resulted in a complete recovery of the CD spectrum from 250 to 2o5 nm to that of the native protein, indicating that the alteration in secondary structure brought about by urea is completely reversible*. This was not TABLE I ELLIPTICITIES AND ESTIMATED HELIX CONTENTS OF NATIVE AND TREATED OVINE F S H E x t r e m e s c a r c i t y of h i g h l y p u r i f i e d m a t e r i a l a l l o w e d o n l y for s i n g l e d e t e r m i n a t i o n s a t t h i s t i m e . T h e v a l u e s o f e l l i p t i c i t i e s a r e a r b i t r a r i l y a s s u m e d t o b e ± 5 ~o o f t h e n o m i n a l v a l u e . T h e v a l u e s a r e c o m p u t e d o n t h e a s s u m p t i o n t h a t t h e m o i s t u r e c o n t e n t o f t h e p r o t e i n is 15 °/o.
(O) MnW (degrees. cm 2. decimole -1) °/o Excess* right hand a-helix*
Sample
210
N a t i v e , p H 8. 3 Urea treated A l k a l i t r e a t e d , p H 12.9 Dialyzed from 8 M urea D i a l y z e d f r o m p H 12.9
~m
220
12 2oo ~ 6oo --i i 500 ~ 575 8 o o o ± 400
* Expressed to the nearest
9 4 5 9 5
n•
9oo ooo ioo 200 800
210
± J= ~ ~ ±
5oo 2oo 250 45 ° 300
3° --25 20
~'lm
220
nm
3° 2o 20 3° 20
5/o.°/
true of the alkali-denatured sample which showed only a partial return of its spectrum to that of the native protein. Ellipticities and estimated helical contents of these dialyzed samples are given in Table I. The CD spectrum of the protein in the region of side-chain absorption is shown in Fig. 2. At pH 8. 3 the spectrum of the native protein shows a weak negative shoulder at 293-294 nm, two overlapping negative bands with apparent peaks at 283 and 273-275 nm, and two additional negative peaks at 267 and 262 nm : the strong negative beginning at about 258 nm is mostly due to the a-helix band at 22o nm. The spectrum of the urea-treated protein is shifted towards less negative ellipticity values from that of the native but shows essentially the same pattern of optically active bands. Upon removal of the urea by dialysis, this portion of the spectrum does not return to that of the native protein, in contrast to the spectrum in the region of peptide-bond absorption. The spectrum at p H I2. 9 shows a complete loss of the negative dichroism exhibited by the native protein from 325 to 275 nm. This is replaced by a fairly strong positive band centered around 25o nm. On dialysis of this material back to pH 8.3 Biochim. Biophys. Aeta, 221 (197 o) i 4 2 - I 4 5
145
SHORT COMMUNICATIONS
this positive peak disappears but with very little reformation of the negative dichroism bands between 260 and 325 nm. As discussed by BEYCHOKz, diehroism in the region from 26o to 325 nm is mainly due to the side chains of the aromatic amino acids, and is very sensitive to the tertiary structure in the immediate vicinity of these residues. The CD spectra of native FSH shows a number of optically active bands in this region. The two negative bands at 267 and 262 nm m a y be tentatively assigned to phenylalanine by comparison with the CD spectra of N-benzyloxycarbonyl-L-phenylalanyl-L-phenylalanine in lOO% ethylene glycoF. The two negative bands at 273-275 and 283 nm, and the negative shoulder at 295 nm m a y be due either to tyrosine, tryptophan or possibly both. The positive peak at 25 ° n m seen at pH 12. 9 is ahnost certainly due to ionized tyrosine residues2, 8. These studies indicate that between one-fourth and one-third of the amino acid residues in FSH are in an a-helical conformation. This secondary structure m a y be partially denatured by treatment with either strong alkali or 8 M urea. While strong alkali appears to bring about an irreversible loss of secondary structure, the loss of a-helix caused by urea treatment is completely reversible. Both treatments produce alterations in the tertiary structure of the molecule as shown by the altered environments of the aromatic residues. However, it should be noted that the effect of urea on the tertiary structure is both less extensive and more nearly reversible than that of alkali. We thank Professor C. H. Li for suggesting this study and for his advice. The work was supported in part by the U.S. Public Health Service Grant A-6o97 from the National Institute of Arthritis and Metabolic Diseases and the Geffen Foundation. One of us (H.P.) is a Career Development Awardee, Institute of General Medical Science, U.S. Public Health Service.
Hormone Research Laboratory, University of California, San Francisco, Cahf. (U.S.A.) I 2 3 4 5 6 7 N
MARGARETA EKBLAD
THOMAS A. BEWLEY HAROLD PAPKOFF
B. JIRGEXSONS, Arch. Biochem. Biophys., 91 (196o) 123. S. BEYCHOK, Science, 154 (1966) 1288. M. EKBLAD, H. PAPKOFF AND C. H. LI, to be published. H. PAPKOFF, D. GOSPODAROWICZ AND C. H. LI, Arch. Biochem. Biophys., 12o (1967) 434. T. A. BEWLEY AND C. H. LI, Arch. Biochem. Biophys., 138 (197 o) 338. G. HOLZWORTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. I. WEINRYB AND R. F. STEINER, Arch. Biochem. Biophys., 131 (1969) 263. N. S. SIMMONS AND A. N. GLAZER, J. Atn. Chem. Soc., 89 (1967) 5o4 o.
Received May I9th, 197o Biochim. Biophys. Acta, 22i (197 o) 142-145