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Reversible denaturation of rabbit-muscle lactate dehydrogenase
391
SHORT COMMUNICATIONS
where [M(2)] is the molecular rotation, connected to specific rotation by the equation M
de is the ellipticity, 40 is [M] ~ [a] 1o~ (M is molecular weight), [0 °] = 2.3 ( ~ ) the wavelength of the absorption maximum, and A 0 is the half-width of the absorption band, equal to 30 m# for the pyridoxylidene form of the enzyme and to 20 m/~ for the pyridoxamine form. It is evident from Fig. 2 that the positions of maxima in the calculated anomalous optical rotatory dispersion curves coincide with those in experimental curves from refs. I and 2, and the amplitudes are of the same order. Thus, it is evident that the measurement of circular dichroism in the absorption bands of protein-bound coenzymes can provide valuable information about the active sites of enzymes, and that the circular dichroism method is much more convenient for such investigations than the spectro-polarimetric study of optical rotation.
Institute of Radiobiology and Physico-Chemical Biology, Academy of Sciences of the USSR, Moscow, (USSR)
Y u . N. BREUSOV
V. I. IVANOV M. YA. KARPEISKY Yu. V. MOROZOV
Y o . M. TORCHINSK¥ AND L. G. KORENEVA, Bzokhimza, 28 (1963) lO87. 2 p. FASELLA AND G. HAMMES, Biochemzstry, 3 (1964) 53 o3 W. T. J~NKINS AND I. W. SIZER, J. Biol. Chem., 234 (1959) 1179. 4 0 . L. POLYANOVSKY, B~okh~mza, 27 (1962) 737. 5 W. T. JENKINS AND I. W. SIZER, J. Biol. Chem., 235 (196o) 62o. M. YA. KARPEISKY AND YU. ~NT.BREUSOV, Biokh~mia, in the press. C. DJERASSI, Optzcal Rotatory Dispersion, McGraw-Hill, New York, 196o, p. 165. z
Recieved July 27th, 1964 Biochim. Biophys. Acta, 92 (1964) 388-391
sc 63o69
Reversible denaturation of rabbit-muscle lactate dehydrogenase Reversible disaggregation (or, at least, hybridization) of the tetramers of lactate dehydrogenase has been carried out by freezing in I M NaCI (ref. I). Under these conditions the tertiary conformations of the monomeric subunits of lactate dehydrogenase were apparently maintained. However, from the point of view of the capacity of amino acid sequences to determine unique tertiary conformations, it is necessary to ascertain whether the subunit polypeptide chains of lactate dehydrogenase can be renatured after complete unfolding. Initial results obtained in experiments with beef lactate dehydrogenase have been in apparent contradiction to the theory ~ that the higher orders of structure in a native protein are determined thermodynamically by the amino acid sequence of its polypeptide chain(s); after exposure of lactate dehydrogenase to concentrated solutions of urea or guanidine • HCI functional enzyme could not be reconstituted 3. Factors such as protein concentration, prevention of adsorption of protein to glassware, and protection o f - S H vulnerable to oxidation (such as exist in lactate dehydrogenase 4) are of critical importance during renaturation in vitroS-L We undertook, therefore, in experiments in which these factors were controlled, to reopen the Biochzm. Bzophys. Acta, 92 (1964) 391-394
3!)2
SHC)RT COMMUNI(
ATI()N.~
question of whether completely (lisaggregated and unfolded lactate dehydrogenase could be renatured. Preparations (,f rabbit mn.--(h, lactate dehydrogenase (E(" I.I 1.27) ((1brained from Mann Research 1.aboratorle>, I.()t No. 1148, and Sigma Chemical ('()mpany, Lot No. 73B-I84O ) were unfolded (denatured) at a protein concentrati(m ()f o.85 mg/ml b y t r e a t m e n t with either Io.5 M urea ()r 5 M guanidine. HC1 at 24 ~ in o.o 5 M p()tassium phosphate huffer (pH 7.4t, contaimng o.12 M [J mercapt()ethanol. Such treatment causes complete dlssocmtu)n of tke lactate dehydrogenase tetramer> /a.~ judged by a change in sedimentation constant), produces maxm~al diaruption ()f the tertiary conformation of the mt)n()n~eric .~ubunits (as iudged hy changes u/ optical rotation, fluorescence intensity and accessihilitv ()f -SH to titration) and results in c()mt)h.te loss of enzymic actix atv a,s. The denatured pr()tein wa> dih~ted 2()o-l~ld with o.I M p(,tas.%iunl t~host)hate huffer (pH 7.4), c()ntaining fi-mercat)tl)ethan,)l (3.8 7.(~ raM) and, in most ca.~e.~, bovine serum albumin (o I mg/ml) At higher ('()ncentrations precipitati(m occurred, presumably because ()f agglegati-n ,)f the unfl)lded lactate dehydrogenase chain.-. The protein was incubated fl)r 4 h at 24", and the re.%ultant clear soluti()n wa< then concentrated 5o-fold l)v v a c u u m dialv.q.- against the same buffer, ('(mtanung/;:-mercapt,)ethanol U . q 3 .~ raM). The >mall a m o u n t (if precipitate that formed wa.%removed b y centrifugati()n, and the activit\' (if the renatured material was as.~ayed 'q. Renaturati(m was t h u , carried ,)ut as a tw(,-stage proces.%: (I) the denatured mon(,mers were allowed to ref()ld at v('ry high dilution, using albumin or siliconlzation ()f the glas~ t() prevent loss ()f pr()tein at the gla~s-liquid interface and [Lmercaptoethanol t,) prevent oxidation ()f tilt' SH group.~ : and {2) the dilute s()lntion (if refl>lded m()nomers wax concentrated in ()rder to induce reaggregatl()n i n t . tetramer.~. By thi~ procedure, moderately good recoveries of activity (I 7 34'>,,) were ()btained (Tat>le I), \Vhen, m addition, the concentration ()f the/#mercapt<)etlaan(ll was reduced (.~ee Tabh' 1) the yield waa ahnost douhled, .~ugge.-tipg that at the hi~laer concentrations the /Lmercaptoethan()l partmlly inhibited rea('ti% at it )n, l><)s.qbly through the f()rmati(m t)f mixed disulfides with the protein - S H groups. The maximal recovery ()f enzyme activity was (~o°. 1)t that tl,,v,ueticallv p<)~,il~le, k'Vhen native lactate dehydr()genase was simply diluted and rec(mcentrated, with()ut exposure tt) urea or gnanidine" H('I, the yield of native enz 5 nw was again ()()(?,, anti a small a m o u n t of precipitate formed The reason for thi~ 111b~()f pr()tein and activity is n()t vet underst()l)d. "1" \ I I L E I ('()ND1TI()N`%
AND
R 1 4 % U L 1".% O F
RI;NATURATION
()1; L \ ( ~1 \~I E I ) E H Y D R O G E N
fl-M~ rcaptocthanol ( nfoldtn~ ~t~('n:
U r e a (~o. 5 M! U r e a ( l O 5 M) ( ; u & n l d i n c • l IC1 (% M ) Guanldme. tit I (5 5I) l , r c a ( t o 5 M) I i r e a ( l o 5 MI None
l~roh,cttT,c
a~nt
.%hcomzatum All)umm \ll)umm \lbumm .\lt)umul Slhcomzatlon .\lbumln
omen dtmn~
fl Mercaptoetham,l
\Sl r
") whl
conch, after
( % .!
"'rcfoldo~"
vacuum d~aly~ls
,)r~tnal
(ram)
t raM)
act~vttv )
7 I, 7 (,
3.~; 3 ~
l7 3I
~)
3 S
3
i i u I
.~ t i,o 5 ~, (~o
-
.3 S 3 .x 3 S 3 S
Hz,)ch~m.
~ () ,) q
Bwphvs
ttla.
t
o e (I()(,4) 3 9 t
304
SHORT COMMUNICATIONS
393
i!l J ÷
msoz~$
'=3
4
Fig. I. Electrophoresls of native and renatured lactate dehydrogenase m s t a r c h gel. L a c t a t e dehydrogenase was unfolded by t r e a t m e n t with 5 M g u a m d i n e . H C 1 and r e n a t u r e d as described m the text. B o t h the r e n a t u r e d material and native lactate dehydrogenase were subjected to electrophoresis in s t a r c h gel, using a T r l s - b o r a t e buffer s y s t e m (pH 8.7)11,13.
Samples of native and renatured lactate dehydrogenase were examined b y electrophoresis in starch gels 1°, a method sensitive to both charge and molecular size. The gels were run in the discontinuous buffer system of POULIK11 and were then sliced and stained for lactate dehydrogenase activity 12. The results are shown in Fig. I. Renatured, diluted-reconcentrated, and native lactate dehydrogenase all gave identical patterns, each containing the various lactate dehydrogenase isozymes in the same proportions. Splitting of the bands was most notable in isozymes 3 and 4 (ref. 13). Enzymic activity, relative to absorbancy at 280 m/~, was the same for both native and renatured lactate dehydrogenase, indicating that the portion of lactate dehydrogenase that remained soluble after renaturation was fully active. This is supported by the observation that no inactive protein band was detectable by electrophoresis when gels were stained for protein 1°. The results of the present study with lactate dehydrogenase are fully compatible with the theory that amino-acid sequence is the major determinant of the threedimensional structure of a protein, and that disulfide bonds (of which lactate dehydrogenase has none) per se are not essential for guiding the folding of polypeptide chains to the corresponding native proteins. Furthermore, these conclusions now appear to be valid not only for protein multimers containing chains of one type, such as enolase and aldolase 14-~8, but also for multimers containing two different polypeptide Biochim. Biophys. Acta, 92 (1964) 391-394
394
SHORT COMMUNI('ATI( )N.~
chains, s u c h as l a c t a t e d e h y d r o g e n a s e i s o z y m e s 2, 3 a n d 4. T h e i m p o r t a n c e of prev e n t i n g c h e m i c a l " d a m a g e " (such ab o x i d a t i o n o f - S H groups) a n d <)f phy.,mal ~-ider e a c t i o n s (such as a d s o r p t i o n o f p r o t e i n s to gla~s) m u s t a g a i n be e m p h a s i z e d , as m u s t t h e n e c e s s i t y for high d i l u t i o n of p r o t e i n d u r i n g t h e first s t a g e of t h e r e n a t u r a t i o n proces~ in o r d e r to p r e v e n t i n t e r m o l e c u l a r a g g r e g a t i o n of u n f o l d e d polypet~tide chains. L a s t l y , it s h o u l d be k e p t in m i n d t h a t t h e c o n d i t i o n s of r e n a t u r a t i o n in vztro are, at best, o n l y a c r u d e a p p r o x i m a t i o n of t h e i n t r a c e l l u l a r e n v i r o n m e n t at t h e sit(, ~f p r o t e i n .~ynthesis. Therefl)re, failure to r e n a t u r e p r o t e i n s zn vitro bhould nl~t, m it~01f, be i n t e r p r e t e d as e v i d e n c e for t h e e x i s t e n c e <>f special protein-fi,ldmfg n w c h a n i s m ~ in vivo.
W e wish to ext>re~.~ o u r a p p r e c i a t i o n to Dr. E. S VESEIA. fl~r perfi~rming t h e s t a r c h - g e l e l e c t r o p h o r e s e s r e p o r t e d in t h i s c o m m u n i c a t i o n . ('HARLES ,]. ITPbTEIN ~IARGAIlET ~[. {'ARTEt,~ ROBERT F. (~)I,I)I~F,I¢(;ER
Laboratory o f Chemical B w l o o ' , N a t i o n a l I n s t i t u t e o f .4 rthritis and Metabolic Diseascs, N a t i o m d I n s t i t u t e s o f Health, Bethesda, M d . ( U . S . A . )
1 C. L. ~IARKERT, Sctence, ~4o (19o3) 1329 C B .~NFINSEN, Brookkaven @,rap I3ml., 15 (191,2) IS 4 3E
~tPPELA AND I'. L ~'[ARKERT, P, locke~l. B~ophys. Res. C'ommun , l, (i()t~l) ~Tt.
H. A. ZONDAt;, Science, 142 (1903) 905 5 R. SHAPIRA, Bwckem. Bwphys. Res. Commun., t (i059) 231) ('. J. EPSTEIN, R F (~;OLDBERGER, 1). ~I Xt'QUNG XND ('. t{. \NFINSEN, .tYl'k B z o c k e m
Btopkys.,
Suppl., I (1902) 223. 7 R . F. GOLDBERGER, C J FPSTEIN XND (' 1{. '\NFINSEN, ,l Btol Chem., 8 G. 1)I SABATO, A. ])ESCE AND N. (). I'~APLAN, I?{lOChl~ll Btophys..4cla, 9 F. lkUBO\VITZ AND I'. ()TT, P, w c h e m . Z . 3 t 4 (19-13) q4
238 [I003) 1,2,~. 77 (~q031 I 3 3
10 O. SMITHIES, Bwchem. J . 7 ~ (1950) 585 n M. D. I>OULIK, Nalure, tSo (1957) 1477 In" E . S VESFLL, ,qttIl. N . Y . . 4 c a d Set . ')t (lqt,i) s77. 18 I). j . FRITZ AND N. B. JACOBBON, Nclence, I4O (tO03) 04
t~ E. \V \VESTHECD, 1.. BI'TI.~tR AND 1' 1). HOYER, t3u,ckemtstrv, 2 (i903) 927. is \V. C. DEAL, \V. J. RUTT~':R, V MAsSI'V XND I'L E. V~N HOLDE, Biochem Bzophvs men., io (1963) 4'0 16 E . STELL\VAGEN ~XNI) ~1. 1( ~CHACHM,\N, Htochelntslrv,
lee~ ('ore-
I (ic)l)2) iON0.
R e c e i v e d A u g u s t xoth, 1964 13~ock~m. Bu)phys, 3cta ~2 11,4o4) 391 304
SHORT COMMUNICATIONS
where [M(2)] is the molecular rotation, connected to specific rotation by the equation M
de is the ellipticity, 40 is [M] ~ [a] 1o~ (M is molecular weight), [0 °] = 2.3 ( ~ ) the wavelength of the absorption maximum, and A 0 is the half-width of the absorption band, equal to 30 m# for the pyridoxylidene form of the enzyme and to 20 m/~ for the pyridoxamine form. It is evident from Fig. 2 that the positions of maxima in the calculated anomalous optical rotatory dispersion curves coincide with those in experimental curves from refs. I and 2, and the amplitudes are of the same order. Thus, it is evident that the measurement of circular dichroism in the absorption bands of protein-bound coenzymes can provide valuable information about the active sites of enzymes, and that the circular dichroism method is much more convenient for such investigations than the spectro-polarimetric study of optical rotation.
Institute of Radiobiology and Physico-Chemical Biology, Academy of Sciences of the USSR, Moscow, (USSR)
Y u . N. BREUSOV
V. I. IVANOV M. YA. KARPEISKY Yu. V. MOROZOV
Y o . M. TORCHINSK¥ AND L. G. KORENEVA, Bzokhimza, 28 (1963) lO87. 2 p. FASELLA AND G. HAMMES, Biochemzstry, 3 (1964) 53 o3 W. T. J~NKINS AND I. W. SIZER, J. Biol. Chem., 234 (1959) 1179. 4 0 . L. POLYANOVSKY, B~okh~mza, 27 (1962) 737. 5 W. T. JENKINS AND I. W. SIZER, J. Biol. Chem., 235 (196o) 62o. M. YA. KARPEISKY AND YU. ~NT.BREUSOV, Biokh~mia, in the press. C. DJERASSI, Optzcal Rotatory Dispersion, McGraw-Hill, New York, 196o, p. 165. z
Recieved July 27th, 1964 Biochim. Biophys. Acta, 92 (1964) 388-391
sc 63o69
Reversible denaturation of rabbit-muscle lactate dehydrogenase Reversible disaggregation (or, at least, hybridization) of the tetramers of lactate dehydrogenase has been carried out by freezing in I M NaCI (ref. I). Under these conditions the tertiary conformations of the monomeric subunits of lactate dehydrogenase were apparently maintained. However, from the point of view of the capacity of amino acid sequences to determine unique tertiary conformations, it is necessary to ascertain whether the subunit polypeptide chains of lactate dehydrogenase can be renatured after complete unfolding. Initial results obtained in experiments with beef lactate dehydrogenase have been in apparent contradiction to the theory ~ that the higher orders of structure in a native protein are determined thermodynamically by the amino acid sequence of its polypeptide chain(s); after exposure of lactate dehydrogenase to concentrated solutions of urea or guanidine • HCI functional enzyme could not be reconstituted 3. Factors such as protein concentration, prevention of adsorption of protein to glassware, and protection o f - S H vulnerable to oxidation (such as exist in lactate dehydrogenase 4) are of critical importance during renaturation in vitroS-L We undertook, therefore, in experiments in which these factors were controlled, to reopen the Biochzm. Bzophys. Acta, 92 (1964) 391-394
3!)2
SHC)RT COMMUNI(
ATI()N.~
question of whether completely (lisaggregated and unfolded lactate dehydrogenase could be renatured. Preparations (,f rabbit mn.--(h, lactate dehydrogenase (E(" I.I 1.27) ((1brained from Mann Research 1.aboratorle>, I.()t No. 1148, and Sigma Chemical ('()mpany, Lot No. 73B-I84O ) were unfolded (denatured) at a protein concentrati(m ()f o.85 mg/ml b y t r e a t m e n t with either Io.5 M urea ()r 5 M guanidine. HC1 at 24 ~ in o.o 5 M p()tassium phosphate huffer (pH 7.4t, contaimng o.12 M [J mercapt()ethanol. Such treatment causes complete dlssocmtu)n of tke lactate dehydrogenase tetramer> /a.~ judged by a change in sedimentation constant), produces maxm~al diaruption ()f the tertiary conformation of the mt)n()n~eric .~ubunits (as iudged hy changes u/ optical rotation, fluorescence intensity and accessihilitv ()f -SH to titration) and results in c()mt)h.te loss of enzymic actix atv a,s. The denatured pr()tein wa> dih~ted 2()o-l~ld with o.I M p(,tas.%iunl t~host)hate huffer (pH 7.4), c()ntaining fi-mercat)tl)ethan,)l (3.8 7.(~ raM) and, in most ca.~e.~, bovine serum albumin (o I mg/ml) At higher ('()ncentrations precipitati(m occurred, presumably because ()f agglegati-n ,)f the unfl)lded lactate dehydrogenase chain.-. The protein was incubated fl)r 4 h at 24", and the re.%ultant clear soluti()n wa< then concentrated 5o-fold l)v v a c u u m dialv.q.- against the same buffer, ('(mtanung/;:-mercapt,)ethanol U . q 3 .~ raM). The >mall a m o u n t (if precipitate that formed wa.%removed b y centrifugati()n, and the activit\' (if the renatured material was as.~ayed 'q. Renaturati(m was t h u , carried ,)ut as a tw(,-stage proces.%: (I) the denatured mon(,mers were allowed to ref()ld at v('ry high dilution, using albumin or siliconlzation ()f the glas~ t() prevent loss ()f pr()tein at the gla~s-liquid interface and [Lmercaptoethanol t,) prevent oxidation ()f tilt' SH group.~ : and {2) the dilute s()lntion (if refl>lded m()nomers wax concentrated in ()rder to induce reaggregatl()n i n t . tetramer.~. By thi~ procedure, moderately good recoveries of activity (I 7 34'>,,) were ()btained (Tat>le I), \Vhen, m addition, the concentration ()f the/#mercapt<)etlaan(ll was reduced (.~ee Tabh' 1) the yield waa ahnost douhled, .~ugge.-tipg that at the hi~laer concentrations the /Lmercaptoethan()l partmlly inhibited rea('ti% at it )n, l><)s.qbly through the f()rmati(m t)f mixed disulfides with the protein - S H groups. The maximal recovery ()f enzyme activity was (~o°. 1)t that tl,,v,ueticallv p<)~,il~le, k'Vhen native lactate dehydr()genase was simply diluted and rec(mcentrated, with()ut exposure tt) urea or gnanidine" H('I, the yield of native enz 5 nw was again ()()(?,, anti a small a m o u n t of precipitate formed The reason for thi~ 111b~()f pr()tein and activity is n()t vet underst()l)d. "1" \ I I L E I ('()ND1TI()N`%
AND
R 1 4 % U L 1".% O F
RI;NATURATION
()1; L \ ( ~1 \~I E I ) E H Y D R O G E N
fl-M~ rcaptocthanol ( nfoldtn~ ~t~('n:
U r e a (~o. 5 M! U r e a ( l O 5 M) ( ; u & n l d i n c • l IC1 (% M ) Guanldme. tit I (5 5I) l , r c a ( t o 5 M) I i r e a ( l o 5 MI None
l~roh,cttT,c
a~nt
.%hcomzatum All)umm \ll)umm \lbumm .\lt)umul Slhcomzatlon .\lbumln
omen dtmn~
fl Mercaptoetham,l
\Sl r
") whl
conch, after
( % .!
"'rcfoldo~"
vacuum d~aly~ls
,)r~tnal
(ram)
t raM)
act~vttv )
7 I, 7 (,
3.~; 3 ~
l7 3I
~)
3 S
3
i i u I
.~ t i,o 5 ~, (~o
-
.3 S 3 .x 3 S 3 S
Hz,)ch~m.
~ () ,) q
Bwphvs
ttla.
t
o e (I()(,4) 3 9 t
304
SHORT COMMUNICATIONS
393
i!l J ÷
msoz~$
'=3
4
Fig. I. Electrophoresls of native and renatured lactate dehydrogenase m s t a r c h gel. L a c t a t e dehydrogenase was unfolded by t r e a t m e n t with 5 M g u a m d i n e . H C 1 and r e n a t u r e d as described m the text. B o t h the r e n a t u r e d material and native lactate dehydrogenase were subjected to electrophoresis in s t a r c h gel, using a T r l s - b o r a t e buffer s y s t e m (pH 8.7)11,13.
Samples of native and renatured lactate dehydrogenase were examined b y electrophoresis in starch gels 1°, a method sensitive to both charge and molecular size. The gels were run in the discontinuous buffer system of POULIK11 and were then sliced and stained for lactate dehydrogenase activity 12. The results are shown in Fig. I. Renatured, diluted-reconcentrated, and native lactate dehydrogenase all gave identical patterns, each containing the various lactate dehydrogenase isozymes in the same proportions. Splitting of the bands was most notable in isozymes 3 and 4 (ref. 13). Enzymic activity, relative to absorbancy at 280 m/~, was the same for both native and renatured lactate dehydrogenase, indicating that the portion of lactate dehydrogenase that remained soluble after renaturation was fully active. This is supported by the observation that no inactive protein band was detectable by electrophoresis when gels were stained for protein 1°. The results of the present study with lactate dehydrogenase are fully compatible with the theory that amino-acid sequence is the major determinant of the threedimensional structure of a protein, and that disulfide bonds (of which lactate dehydrogenase has none) per se are not essential for guiding the folding of polypeptide chains to the corresponding native proteins. Furthermore, these conclusions now appear to be valid not only for protein multimers containing chains of one type, such as enolase and aldolase 14-~8, but also for multimers containing two different polypeptide Biochim. Biophys. Acta, 92 (1964) 391-394
394
SHORT COMMUNI('ATI( )N.~
chains, s u c h as l a c t a t e d e h y d r o g e n a s e i s o z y m e s 2, 3 a n d 4. T h e i m p o r t a n c e of prev e n t i n g c h e m i c a l " d a m a g e " (such ab o x i d a t i o n o f - S H groups) a n d <)f phy.,mal ~-ider e a c t i o n s (such as a d s o r p t i o n o f p r o t e i n s to gla~s) m u s t a g a i n be e m p h a s i z e d , as m u s t t h e n e c e s s i t y for high d i l u t i o n of p r o t e i n d u r i n g t h e first s t a g e of t h e r e n a t u r a t i o n proces~ in o r d e r to p r e v e n t i n t e r m o l e c u l a r a g g r e g a t i o n of u n f o l d e d polypet~tide chains. L a s t l y , it s h o u l d be k e p t in m i n d t h a t t h e c o n d i t i o n s of r e n a t u r a t i o n in vztro are, at best, o n l y a c r u d e a p p r o x i m a t i o n of t h e i n t r a c e l l u l a r e n v i r o n m e n t at t h e sit(, ~f p r o t e i n .~ynthesis. Therefl)re, failure to r e n a t u r e p r o t e i n s zn vitro bhould nl~t, m it~01f, be i n t e r p r e t e d as e v i d e n c e for t h e e x i s t e n c e <>f special protein-fi,ldmfg n w c h a n i s m ~ in vivo.
W e wish to ext>re~.~ o u r a p p r e c i a t i o n to Dr. E. S VESEIA. fl~r perfi~rming t h e s t a r c h - g e l e l e c t r o p h o r e s e s r e p o r t e d in t h i s c o m m u n i c a t i o n . ('HARLES ,]. ITPbTEIN ~IARGAIlET ~[. {'ARTEt,~ ROBERT F. (~)I,I)I~F,I¢(;ER
Laboratory o f Chemical B w l o o ' , N a t i o n a l I n s t i t u t e o f .4 rthritis and Metabolic Diseascs, N a t i o m d I n s t i t u t e s o f Health, Bethesda, M d . ( U . S . A . )
1 C. L. ~IARKERT, Sctence, ~4o (19o3) 1329 C B .~NFINSEN, Brookkaven @,rap I3ml., 15 (191,2) IS 4 3E
~tPPELA AND I'. L ~'[ARKERT, P, locke~l. B~ophys. Res. C'ommun , l, (i()t~l) ~Tt.
H. A. ZONDAt;, Science, 142 (1903) 905 5 R. SHAPIRA, Bwckem. Bwphys. Res. Commun., t (i059) 231) ('. J. EPSTEIN, R F (~;OLDBERGER, 1). ~I Xt'QUNG XND ('. t{. \NFINSEN, .tYl'k B z o c k e m
Btopkys.,
Suppl., I (1902) 223. 7 R . F. GOLDBERGER, C J FPSTEIN XND (' 1{. '\NFINSEN, ,l Btol Chem., 8 G. 1)I SABATO, A. ])ESCE AND N. (). I'~APLAN, I?{lOChl~ll Btophys..4cla, 9 F. lkUBO\VITZ AND I'. ()TT, P, w c h e m . Z . 3 t 4 (19-13) q4
238 [I003) 1,2,~. 77 (~q031 I 3 3
10 O. SMITHIES, Bwchem. J . 7 ~ (1950) 585 n M. D. I>OULIK, Nalure, tSo (1957) 1477 In" E . S VESFLL, ,qttIl. N . Y . . 4 c a d Set . ')t (lqt,i) s77. 18 I). j . FRITZ AND N. B. JACOBBON, Nclence, I4O (tO03) 04
t~ E. \V \VESTHECD, 1.. BI'TI.~tR AND 1' 1). HOYER, t3u,ckemtstrv, 2 (i903) 927. is \V. C. DEAL, \V. J. RUTT~':R, V MAsSI'V XND I'L E. V~N HOLDE, Biochem Bzophvs men., io (1963) 4'0 16 E . STELL\VAGEN ~XNI) ~1. 1( ~CHACHM,\N, Htochelntslrv,
lee~ ('ore-
I (ic)l)2) iON0.
R e c e i v e d A u g u s t xoth, 1964 13~ock~m. Bu)phys, 3cta ~2 11,4o4) 391 304