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Temperature and pH dependent changes of immunoglobulin G structure
Biochimica et Biophysica Acta, 386 (1975) 155-167
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36955 T E M P E R A T U R E A N D pH D E P E N D E N T C H A N G E S OF IMMUNOGLOBULIN G STRUCTURE
VLADIMIR P. ZAV'YALOV, GERMAN V. TROITSKY, ALEXANDER P. DEMCHENKO and IGOR V. GENERALOV Department of Biochemistry and Department of Physics, Crimean Medical Institute, Simferopol, Institute of Biochemistry, Kiev (U.S.S.R.)
(Received July 9th, 1974) (Revised manuscript received October 14th, 1974)
SUMMARY 1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning micrccalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 °C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 °C in the narrow temperature interval (3-4 °C). 4. The "acid state" is not accompanied by S-like transition at 25-35 °C. The thermal denaturation of the "acid state" occurs at 54 °C in the wide temperature interval (8-9 °C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 °C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.
INTRODUCTION We reported previously [1, 2] the existence of a reversible conformational transition of IgG fractions with different isoelectric points at pH 7.3-7.4 and the temperatures between 27 ± 2 and 38 :k 2 °C. The temperature dependent changes disappeared at pH values lower than 6.0 and higher than 8.5. A transition occurring at a high temperature was accompanied by a decrease in levorotation in the visible
156 region of the spectrum, intensification of the Cotton-effect minimum at the 228-225 nm region and an increase in the negative value of the parameter of the Moffit equation b0. A similar transition was observed for the myeloma IgG. The thermal perturbation difference spectra investigations showed an alteration in the asymmetric environment of tyrosine residues within the transition interval. These data and the early data [3-5] on lgG from human, bovine and horse blood serum showed that these changes occurring in physiological conditions are a very general IgG molecular property. In this paper we investigated the temperature and pH functions of the myeloma IgG (K) conformation by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and by difference adiabatic scanning microcalorimetry. The thermal difference spectra alterations as a result of antigen-antibody reaction were studied. MATERIALS AND METHODS
Purification of myeloma IgG. The blood serum was obtained from a hospital patient diagnosed as having multiple myeloma and the protein was precipitated with (NH4)zSO4 in the ratio 17.5 g of salt to 100 ml of serum. Purification was achieved by ion-exchange column chromatography on DEAE-cellulose (Whatman, England) using a gradient of increasing ionic strength (0.005-0.0175 M sodium phosphate) and decreasing pH (8.0-6.3). The absorbance of the fractions was measured at 280 nm. The major component was eluted with 0.01 M sodium phosphate buffer, pH 7.5. Further purification was achieved by gel filtration on Sephadex G-200. Typing. The myeloma immunoglobulin obtained from chromatography was typed by immunodiffusion in agar as G (K), using commercially prepared antisera to IgG, IgA, IgM, K- and L-chains (Behringwerke, G.F.R.). Homogeneity. The myeloma IgG (K) was submitted to electrofocusing in a carrier Ampholine using the Valmet horizontal apparatus of our own modification [6, 7]. Two fractions were obtained with isoelectric points of 6.15 and 6.5. The major component had an isoelectric point of 6.15. Specifically purified anti-DNS* antibody. Specifically purified rabbit anti-DNS antibody was isolated individually from the same immune serum using the same procedure as described previously [8]. Optical rotatory dispersion and circular diehroism. The optical rotatory dispersion over the wavelength range from 300 to 210 nm was investigated in a Jasco ORD/UV-5 spectrophotopolarimeter in a i mm long controlled-temperature cell. with protein concentrations from 0.05 to 0.07 ~ . CD over the wavelength range from 260 to 205 nm was investigated in a Jasco ORD/CD-20 spectropolarimeter in a 2 mm long controlled-temperature cell, with protein concentrations from 0.04 to 0.05~. The ORD over the wavelength range from 600 to 300 nm was investigated in a polarimeter with a two channel system of accumulation [9]. The iodine lamp was used as the source of light. The monochromatic light was obtained using the monochromator in combination with the interference light filters. The optical path was * DNS, l-dimethylaminonaphtalene-5-sulfonyl.
157 10 cm and the protein concentration was 0.4--0.8 ~. Purification with 0.22 nm millipore filters made it possible to obtain high-transparency solutions and reduce the error in the rotation-angle determinations to ± 0.0005 ° for the protein concentrations in question. Thermal perturbation difference spectra. The spectra were investigated in a Hitachi-124 spectrophotometer in 10 mm long controlled-temperature cells with protein concentrations from 0.04 to 0.07 ~ , Part of the spectra was investigated in a Specord UV VIS (Carl Zeiss, G.D.R.) spectrophotometer in 10-mm long controlledtemperature cells with protein concentration 0.106 ~ . Solvent perturbation difference spectra. These spectra were investigated in a Specord UV VIS spectrophotometer in a 10 mm long controlled-temperature tandem cells with protein concentration 0.106~. The perturbants employed in this study were all of spectroscopic grade. Difference adiabatic scanning microcalorimetry. These measurements were made with a DASM-1 [10] microcalorimeter in the thermodinamic laboratory of the Institute of Protein Research (Poustchino). The protein concentration was from 0.1 to 0.3~o. Electrochemical iodination. Selective modification of tyrosine residues was performed with the potentiostatic method of electrochemical iodination [11] in the electrochemical cell with the addition of sodium iodine. The protein concentration was 6.5 mg/ml, E = 545 mV, the volume was 6 ml, the time of the iodination was 20 min. The inorganic forms of iodine were removed using adsorbents described previously [12] and the percentage of combined iodine was determined [12]. The amount of mono-, and diiodtyrosine was determined by difference spectra between non-iodine and iodine protein at pH 13 [13]. pH measurements. Measurements of pH were made on a Radiometer pH meter (Denmark) and pH meter-246 (U.S.S.R.) with a glass electrode. Protein concentration, l~rotein concentration was determined from the absorbance at 280 nm with a Hitachi-124 and a Specord UV VIS spectrophotometer and a factor of 14.1 for a 1 g/100 ml of solution of IgG in 1.0 cm optical path. RESULTS
Exposure of lgG (K) chromophores at 20 °C The solvent perturbation difference spectra of IgG (K) in 20 ~o glycerol and in 2 0 ~ ethylene glycol are presented in Fig. 1A and lB. The curves were obtained at 20 °C in 0.01 M sodium phosphate buffer, pH 7.35. In order to make a quantitative estimate of the number of tyrosine and tryptophan residues exposed to perturbant, Okulov [14] has proposed the following equations: A E293(protein) = aTrp " A e~a
,d ezs7(protein) = aTrp • A E287 ra 0.4 +
(1) bTy r "
A e2M870.6
(2)
where a and b represent the number of tryptophans and tyrosines exposed. The Ae (protein) and Ae M values refer to molar absorption differences for the protein and
158 -0.01
260
280
300
320
3 4 0 ;k i'~m
-0.02
°t
0.02
,
287
i
260
i
2,;o
%f-J ~93-294
h
C ,
--
3~o 7 , ~
Fig. 1. The solvent perturbation difference spectra (A, B) and the thermal perturbation difference spectra (C) of IgG(K). The optical path was 1.0 cm and the protein concentration was 0.106 % (A, B) and 0.071 ~ (C). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The perturbants were 20~ glycerol (A) and 20~ ethylene glycol (B). The solvent perturbation difference spectra were measured at 20 °C. The measurements of the thermal perturbation difference spectra was made at the following conditions: The control cell temperature was 13.2 °C, the sample 23.8 °C.
the model compounds (56 Tyr and 22 Trp)[14] at the designated wavelengths. Typical results are presented in Table I. Fig. IC shows the thermal perturbation difference spectra of lgG (K) in 0.01 M phosphate buffer, pH 7.35. The control cell temperature was 13.2 °C and that of the sample was 23.8 °C. It can be seen that the spectra have two a b s o r b a n c e m a x i m a at 294 a n d 287 nm. It is k n o w n [15, 16] that the model spectra of tyrosine have a m a x i m u m at 287 n m a n d the model spectra of t r y p t o p h a n have a m i n i m u m at 292-293 nm. The longer wavelengths obtained for these m a x i m a of IgG (K) may
TABLE I EXPOSURE OF IMMUNOGLOBULIN G(K) CHROMOPHORES AT 20 °C Data calculated from the analysis of IgG(K) and the model compounds (56 tyrosine and 22 tryptophan) [14] in each perturbant according to equations (1) and (2) of text. Perturbant
de293" 10 s
,de~3" 103
No. of
/le287" 103
/1e2~7' 103
No. of
(20~)
(protein)
(model compounds)
exposed Trp
(protein)
(model compounds)
exposed Tyr
Ethylene glycol Glycerol
1.05 0.75
7.2 6.9
3 2
1.97 1.51
8.0 7.5
17 14
159 be connected with the definite contribution of the chromophores in the medium of low polarity. As the model system for IgG (K) we used a tyrosine solution in 40% ethylene glycol with a maximum spectra at 288 nm and a tryptophan solution in 60% ethylene glycol with a maximum spectra at 295 nm. In order to make a quantitative estimate of exposed chromophores Demchenko and Zyma [16] proposed the use of the extinction increment: (3) where 1 = 285-288 nm or 293-295 nm. The extinction increment is not dependent upon the temperature interval and the absolute absorption for Ezso 5 3. The extinction increment is dependent upon the polarity of the chromophore environment. The calculation of the number of tyrosine and tryptophan residues exposed to solvent was made using the following equations : o/oexposed Tyr = -
% exposed
Trp = -
Xii,” TYI x288
Xii,”
(5)
TIP
X295 at the temperature
intervals
IO-27 and 38-50 “C. The results are presented
in Table II.
TABLE II EXPOSURE t “C
OF IMMUNOGLOBULIN
Chromophores
G(K) CHROMOPHORES Chromophores
Protein I max
&so
A& -.104
X1.103
Ethylene glycol
0.782 0.524 0.193 0.427
40 60 40 60
At IO-27 38-50
Tyr Trp Tyr Trp
Temperature
287 295 287 295
dependent
0.368 0.632 0.368 0.632
2.9 3.3 0.71 2.7
A,,,,.
__ x;hr. lo3
% Exposed chromophores
No. of exposed chromophores
287 295 287 295
2.1 0.97 2.1 0.97
0.373 0.54 0.092 0.44
19.4 12 4.8 9.7
(%I
changes of IgG (K)
structure
Fig. 2A shows the optical rotation dependence on temperature at 551 nm. The solvent was 0.01 M sodium phosphate buffer, pHb7.35. The measurements were made on the same day of the protein preparation and 1 day and 5 days after preparation. The same protein solution was used for these investigations. Between the experiments the solution was kept in the spectropolarimetric cell at 4 “C. It can be seen that between 25 and 35 “C there were S-like transitions which were accompanied by an increase in the optical rotation. The changes outside the transition interval were observed. After storage the temperature dependence outside the transition range increases and the coefficient of the temperature dependence below the transition interval is equal to the coefficient of the temperature dependence above the transition
160
\
• 1 o 2 x3
O'tO 2b t,E
20
I,~o I I
40
t*~"
t *C
Fig. 2. The temperature function of the optical rotation (A) and the thermal perturbation difference spectra (B). The optical path was 10.0 cm (A), 1.0 cm (B). The protein concentration was 0.394 % (A) and 0.071% (B). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The measurements of the optical rotation (A) were made on the same day of protein preparation (1) and l day (2) and 5 days (3) after preparation. The thermal perturbation difference spectra were investigated at the following conditions: (B) The control cell temperature was 10 °C, the temperature of the sample was changed; (1) 287-288 nm, (2) 294-295 nm.
interval. The temperature dependence of the optical rotation outside the transition interval disappears when the concentration of the sodium phosphate buffer was increased to 0.1 M, but the parameters of the transition investigated are not altered. Fig. 3A shows the O R D curves for IgG (K) at pH 7.5 over the wavelength region from 300 to 210 nm at 20 and 40 °C. It can be seen that at 20 °C the O R D curve has a minimum at 224--225 nm and a shoulder at 228-229 nm. At 40 °C there was only a minimum at 228-229 nm. Fig. 3B shows the thermal perturbation difference spectra for IgG (K). The curves were obtained under the following conditions: (1) The control cell temperature 13.2, the sample 23.9 °C; (2) The control cell temperature 37.2, the sample 47.9 °C; (3) The control cell temperature 13.2, the sample 47.5 °C. The solvent was 0.01 M phosphate buffer, pH 6.9. It can be seen that the spectra below 25 °C and above 35 °C are significantly different, especially at 287 nm. Fig. 2B shows the temperature dependence of A E at 287 and 295 nm. It can be seen that the temperature dependence has an S-like form especially at 287 nm. The observed interval of the S-like transition coincides with the abrupt transition observed with optical rotation. The calculation of the exposed chromophores below and above the temperature transition is presented in Table II. It can be seen that the exposure of tyrosine residues decreases as a result of the temperature transition. We tested this effect by measuring the solvent perturbation difference spectra at 20 and at 40 °C using 20 % ethylene glycol as perturbant. The results of such a treatment are presented in Fig. 3C. It can be seen that the maximum at 287 nm decreases as a result of heating from 20 to 40 °C. We tested the effect on tyrosine residues screening by the electrochemical
161
[a(];~.l-3 O C 225 245 265 ;~nm -1 ........
i
3
-0,01 o
o.o~
B
AE'a~°
'
a~o
'
35o ' 3~o ~ "
- 0,011 0.011
~
=
,
260
C ,
28o
,
p
300
,
=
320 ;~ nm
Fig. 3. The temperature function of the optical rotation dispersion (A), the thermal perturbation difference spectra (B) and the solvent perturbation difference spectra (C) of IgG(K). The optical path was 0.1 cm (A) and 1.0 cm (B, C). The protein concentration was 0.071 ~ (B, (2) and 0.081 ~ (A). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The ORD (A) was measured at 20 °C (1) and at 40 °C (2). The thermal perturbation difference spectra were measured at the following conditions: The control cell temperature was 10 °C (1, 3) and 39 °C (2), the sample 20 °C (1) and 49 °C (2, 3). The solvent perturbation difference spectra (C) were measured at 20 °C (1) and at 40 °C (2). The perturbant was 20 ~ ethylene glycol. i o d i n a t i o n m e a s u r e m e n t s at 20 and 40 °C. T h e results are presented in Table III. It can be seen that at 40 °C the percentage o f c o m b i n e d iodine (at o t h er c o n s t a n t conditions) decreases by twice the value c o m p a r e d with the i o d i n a t i o n at 20 °C. Th e results o f i o d i n a t i o n in the m ix tu r e 1:4 are n o t in c o n t r a d i c t i o n with this conclusion because the a m o u n t o f m o n o i o d i n e tyrosines decreases by 6. We controlled the conf o r m a t i o n changes after the iodination. In a c c o r d a n c e with the C D data the conf o r m a t i o n o f I g G (K) is n o t altered u n d e r these conditions. TABLE III THE RESULTS OF THE ELECTROCHEMICAL IODINATION OF IgG(K) AT 20 AND 40 °C Protein/iodine mixture
20 °C
40 °C
Percentage of combined iodine
Amount of monoiodine Tyr (- 10s ,u/I)
Amount of diiodine Tyr (. l0 s ,u/I)
1 :I
85
--
_
1:2
99
--
_
1:4
83
10.85
3.01
1:8
79
--
_
Percentage of combined iodine
35
48 85 48
Amount of monoiodine Tyr (-105/~/l) --
-1.89 --
Amount of diiodine Tyr (" 10s/~/1) --
-6.72 --
162 We expected a heat effect as a result of the transition investigated, but the microcalorimetry measurements detected only a regular increase of the thermal heat capacity between the temperatures 20 and 50 “C. The pH-dependence
of the temperature
changes of IgG (K)
Fig. 4A shows the optical rotation temperature dependence of IgG (K) at different pH values. Fig. 4B shows the dependence of the temperature transition amplitude on pH. It can be seen that the temperature transition exists only in a narrow interval of pH: from 6.0 to 7.8. The transition amplitude reaches the maximum value at pH 6.9-7.0.
Fig. 4. The pH-function of the temperature changes of IgG(K) using the optical rotation (A, B) and the thermal perturbation difference spectra (C, D). The optical path was 10.0 cm (A, B) and 1.Ocm (C, D) and the protein concentration was 0.394% (A, B) and 0.13 % (C, D). The solvent was 0.01 M sodium phosphate buffer or 0.1 M acetate buffer at correspondent pH values. The measurements of the optical rotation (A) were made at the following pH values: 6.05 (l), 6.1 (2), 6.45 (3), 6.85 (4), 7.35 (5), 7.43 (6), 7.50 (7), 7.75 (8). (B) The pH-function of the amplitude of temperature transition of human myeloma IgG(K) (1) and the isoelectric fractions of IgG from bovine serum with p17.35 (2) and pZ7.95 (3). The pH-function of the thermal perturbation difference spectra (C, D) was measured at the following conditions: The control cell temperature was 23 “C, the sample 45 “C. (C, curve 1). The thermal perturbation difference spectra above pH 6.5. (C, curve 2) The spectra below pH 6.0. (D, curve x) E at 287-288 nm. (D, curve o) E at 294-295 nm.
Fig. 4C shows the temperature perturbation difference spectra of IgG (K) at different pH values. The temperature of the control cell was 23 “C, the sample temperature was 45 “C. The pH-dependence of the absorptivity difference at 287 nm and 295 nm is presented in Fig. 4D. It can be seen that the S-like transition occurs in the narrow interval of pH values (6.5-6.0). The S-like transition occurs in the narrow interval of pH values (6.5-6.0). The S-like transition occurring at lower pH is accom-
163 panied by an increase in the amplitude of the maxima at 287 and 295 nm and by a decrease in the optical rotation transition amplitude. It can be seen (Fig. 5) that the pH-dependence of a "high temperature state" (above 35 °C) is more than the pHdependence of a "low temperature state" (below 25 °C). At the physiological conditions of pH and temperature the exposure of tyrosine and tryptophan residues reaches the minimal value. -
tXE
O
.
O
1
~
(3
o.o1~-
a
v
m-
-
Fig, 5. The pH-function of the thermal perturbation difference spectra. The optical path was 1.0 cm. The protein concentration was 0.071 ~ . The solvent was 0.01 M sodium phosphate buffer or 0.] M
acetate buffer at correspondent pH values. The control cell temperature was 13.5 °C (1, 2) and 37 °C (3, 4), the sample 24.5 °C (1, 2) and 47 °C (3, 4); (1, 3), pH 7.0; (2, 4), pH 5.85. We investigated the thermal stability of the pH-dependent structural states. The results are presented in Fig. 6. It can be seen that the "acid state" does not accomplish the cooperative transition at 25-35 °C. The denaturation of the "acid state" occurs at 54 °C in the wide temperature interval (8-9 °C). After the thermal denaturation Trp residues are fully exposed to solvent and approx. 60 ~ of tyrosine residues are exposed. The "alkaline state" was found to be capable of fully reversible S-like transition at the temperatures between 25 and 35 °C (see Fig. 2B). The heating of IgG (K) from 45 to 63-64 °C is accompanied by the decrease of maxima at 287 and 295 nm. This process is linear and has a sign opposite to that for the thermal denaturation. The denaturation of the "alkaline state" occurs only at 64 °C in the narrow temperature interval (3-4 °C). The denaturation is accompanied by irreversible aggregation. According to the CD data there are no differences between the conformation for "acid" and "alkaline" states.
The thermal perturbation difference spectra of rabbit anti-DNS antibody before and after hapten-antibody reaction Fig. 7 shows the spectra of specifically purified rabbit anti-DNS antibody before and after hapten, antibody reaction. The temperature of the control cell was 17 °C, the sample 37 °C. The solvent was 0.005 M phosphate buffer with 0.15 M NaCI, pH 7.35. The hapten was added in a ratio of 2:1 to the protein. It can be seen that after hapten.antibody reaction the amplitude of the spectra sharply decreases especially at 287 and 295 nm. According to this result about 7 5 ~ of tyrosine and tryptophan residues are buried after hapten-antibody reaction. DISCUSSION According to the results obtained at pH 7.35 and 20 °C approx. 14 tyrosine and 2 tryptophan appear to be accessible to the glycerol; 17 tyrosine residues and
164
o0,I o
,.~
2~o~..
3oo
.~°~=
a / ~-" / / ~ J///7
o.o1~
~.~D"'.,;:/
/ ~ AE 1
ill
/
-oo~
,z_g
I,,,~ ~jr //,4"x
.,q\~ ~,~
/
0.01
"
i ~ ~E~ B
/x" •I
0.02
×2
//..7 /
o.oi
/ ×
/
~I J
x'
40
2b
oo
' t °c "~
Fig. 6. The thermal perturbation difference spectra of IgG(K). The optical path was 1.0 cm and the protein concentration was 0.047 7o. The solvent was 0.01 M sodium phosphate buffer, pH 7.0 (B and C, curve 1) and 0.1 M acetate buffer, pH 4.35 (A and C, curve 2). The temperature of a control cell was 13.6 °C. The temperature of the sample was changed in the following sequence: (A) 19.0 (1), 25 (2), 30.5 (3), 35.3 (4), 39.5 (5), 44.5 (6), 49.3 (7), 54.8 (8), 58.5 (9), 63.1 (10), 67 (11), 71.5 °C (12); (B) 45.5 (l), 48.5 (2), 51.5 (3), 54.5 (4), 57.5 (5), 60.6 (6), 63.6 (7), 66.6 °C (8). zXE
-
0.O1
oi
~10
~
~
2~0
2~0
'
~,~0 ~'~.
0.01 Fig. 7. The changes of the thermal perturbation difference spectra of the rabbit anti-DNS antibodies upon interaction with hapten. The optical path was 1.0 cm and the protein concentration was 0.054 ~ . The solvent was 0.005 M sodium phosphate buffer with 0.15 M NaCl. (1) The spectrum of free antibodies; (2) The spectrum of antibodies uoon interaction with hapten in molar ratio 1:2.
165 3 tryptophan residues are affected by the ethylene glycol and 19-20 tyrosine and 12 tryptophan are exposed according to the thermal perturbation difference spectra. The IgG (K) studied was found to be capable of a fully reversible S-like transition at p H values between 6.5 and 6.0. The changes occurring at lower p H are accompanied by an increase in exposure of the chromophores to solvent. In agreement with the results of the thermal denaturation the cooperativeness and stability of the "acid" state decreases sharply in comparison with the "alkaline state". This effect may be explained by the intramolecular dissociation of the domains due to ionisation of the imidazoles. The pH-changes investigated look very much like the pH-dependent dissociation of the light chain dimer [17]. The "alkaline state" was found to be capable of a fully reversible S-like transition at the temperatures between 25 and 35 °C. The changes occurring at the higher temperatures are accompanied by the screening of 14-15 tyrosine, but the transition is not accompanied by an appreciable heat effect. One of explanations of this contradiction may be the simulation of pseudo cooperativeness by the optical methods used. Another explanation may be the intramolecular compensation of the heat effect. A diagram of the states of IgG (K) is presented in Fig. 8. It can be seen that IgG (K) has some different states in conditions far from denaturation. :
. - _ ' . . ,
-,_."
.
.
. -._-
..'.'."
t *C
60
4O
20 I
i
i
i
i
2
4
6
8
10
pH
Fig. 8. The diagram of states of IgG(K) in the investigated region of pH and temperature values. The shade regions correspond to transition intervals. B, unfolded state; A~, folded state with a maximal screening of chromophores (associated); A~a, folded associated state above 45-50 °C; A~, folded "alkaline" state with a maximal exposure of chromophores (dissociated); A~, folded 'l'acid" state with a maximal exposure of chromophores. N, the region of existence of the native IgG structure at the physiological conditions of warm-blooded animals. Fig. 9 shows the general conformation of lgG (K) Fab fragment. The model is extrapolated as far as structural information is concerned partly from a Fab fragment [18, 19], and partly from a lambda chain dimer [20]. The numbering of amino acid residues in the K-chains is in accordance with alignment No. 50 [21], in the VH domain, in accordance with alignment No. 54 [21] and in the Cm domain, in accordance with alignment No. 55 [21 ]. Aromatic and charged side groups are marked using one letter code or the sign of the charge. The marked side chains are invariant in the K-chains observed. It can be seen that the invariant aromatic side chains are situated in the cavities between the domains and may be good markers for the investigation of the domain interposition. The ionisation of the invariant histidine
166 (+)1S
"rs
~
2
s8
6a
"~
VL
Fig. 9. The general conformation of ]gG(K) Fab fragment. The model is extrapolated as far as structural information is concerned, partly from a Fab fragment [18, ]9], and partly from a lambda chain dimer [20]. The numbering of amino acid residues in K-chains is in accordance with aligment No. 50 [21] and in Cm domain; in accordance with alignment No. 55 [2]], in VH domain; in accordance with alignment No. 54 [21 ]. Aromatic and charged side groups are marked using one letter code or the sign of the charge. The marked side chains are invarJant in K-chains observed.
residues situated in the cavity between the constant and variable domains may cause the pH transition studied. It is very important that a similar effect of about 75 chromophores screening is observed as a result of hapten, antibody interaction. This effect may be explained in the following way. At the temperature of warm-blooded animals (37-38 °C) all the molecules of antibody are in a "high temperature" native state. Only this state has a maximal affinity to the antigen. Thus the antigen stabilizes the "high temperature" native state of antibody. It is interesting that Pilz et al. [22] observed a decrease of the anti-poly(Dalanyl) antibodies volume by 1 0 ~ upon interaction with tetra-D-alanyl hapten. K/iiv/~r~iinen et al. [23] showed that the antigen, antibody interaction is accompanied by a decrease of the mobility of conjugated with histidine residues spin-labels. These investigations are in agreement with our results. The defect of the investigation of myeloma proteins with hapten-binding activity is the selection, in most cases, of specifically binding hapten at room temperatures. Thus one can explain the absence of structural alterations of myeloma proteins upon interaction with hapten [19, 24, 25] because the "low temperature" native state, in this case, is in the state of higher affinity. ACKNOWLEDGEMENTS The authors wish to thank Dr V. N. Kulakov of the Institute of Biophysics (Moscow) for assistance with the electrochemical iodination, Dr Chechinashvili, N.N. of the Institute of Protein Research (Poustchino) for assistance with the microcalorimetry measurements, Dr R. S. Nezlin of the Institute of Molecular Biology
167 (Moscow) for supply of rabbit anti-DNS serum and Dr V. I. Burlev of the Central Institute of Haematology and Blood Transfusion (Moscow) for supply of myeloma serum. The Authors thank Dr P. L. Privalov of the Institute of Protein Research (Poustchino) for helpful discussion of these results. REFERENCES 1 2 3 4 5 6 7 8 9 10
Kirjukhin, I. F., Troitsky, G. V. and Zav’yalov, V. P. (1972) Mol. Biol. 6, 196-200 Troitsky, G. V., Zav’yalov, V. P. and Kirjukhin, I. F. (1973) Biochim. Biophys. Acta 322, 53-61 Zav’yalov, V. P., Kirjukhin, I. F. and Troitsky, G. V. (1970) Mol. Biol. 5, 655-662 Troitsky, G. V., Zav’yalov, V. P. and Kirjukhin, I. F. (1971) Biofizika 16, 785-793 Troitsky, G. V., Zav’yalov, V. P. and Kirjukhin, I. F. (1971) Biochimia 36, 1107-l 114 Valmet, E. (1969) Sci. Tools 16, 8-13 Kirjukhin, I. F. (1972) Bull. Exp. Biol. Med. 74, 120-122 Zagyansky, Yu. A., Nezlin, R. S. and Tumerman, L. A. (1969) Immunochemistry 6, 787-794 Troitsky, G. V. and Generalov, I. V. (1973) Pribori i technika eksperimenta No. 4,261 Privalov, P. L., Makurin, P. C., Kotel’nikov, G. B., Krylov, G. P., Stepanyuk, G. P., Plotnikov, V. V., Koryagin, V. V. and Polpudnikov, V. S. (1972) Abstr. Contr. Papers 4 Int. Biophys. Congr. Moscow 4, 320-321 11 Mojaisky, A. M., Kulakov, V. N. and Stanko, V. I. (1971) Isotopenpraxis 7, 17-20 12 Bagenova, T. L., Mojaisky, A. M., Kulakov, V. N. and Stanko, V. I. (1972) Isotopenpraxis 8, 460-462 13 Klimova, T. P., Kulakov, V. N., Mojaisky, A. M., Bagenova, T. L. and Stanko, V. I. (1973) Stud. biophys. 38, 235-238 14 Okulov, V. I. and Troitsky, G. V. (1969) J. Mol. Biol. 3, 495-506 15 Bello, J. (1970) Biochemistry 9, 3562-3571 16 Demchenko, 0. P. and Zyma, V. L. (1974) Ukr. Biochim. J. 46, 345-350 17 Green, R. W. (1973) Biochemistry 12, 3225-3231 18 Poljak, R. J., Amzel, L. M., Avey, H. P., Chen, B. L., Phizackerley, R. P. and Saul, F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3305-3310 19 Segal, D. M., Padlan, A. E., Cohen, G. H., Rudikoff, S., Potter, M. and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U.S. in press 20 Schiffer, M., Girling, R. L., Ely, K. R. and Edmundson, A. B. (1973) Biochemistry 12,462@-4630 21 Dayhoff, M. (1972) Atlas of Protein Sequence and Structure, Nat. Biomed. Res. Found., Silverspring, U.S.A. 22 Pilz, J., Kratky, O., Light, A. and Sela, M. (1973) Biochemistry 12, 4998-5005 23 Klivlrlinen, A. I., Nezlin, R. S., Lichtenstein, G. I., Misharin, A. Yu. and Volkenstein, M. V. (1973) J. Mol. Biol. 7, 76&768 24 Padlan, E. A., Segal, D. M., Spande, T. F., Davies, D. R., Rudikoff, S. and Potter, M. (1973) Nat. New Biol. 245, 165-167 25 Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M. and Richards, F. F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1427-1430
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 36955 T E M P E R A T U R E A N D pH D E P E N D E N T C H A N G E S OF IMMUNOGLOBULIN G STRUCTURE
VLADIMIR P. ZAV'YALOV, GERMAN V. TROITSKY, ALEXANDER P. DEMCHENKO and IGOR V. GENERALOV Department of Biochemistry and Department of Physics, Crimean Medical Institute, Simferopol, Institute of Biochemistry, Kiev (U.S.S.R.)
(Received July 9th, 1974) (Revised manuscript received October 14th, 1974)
SUMMARY 1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning micrccalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 °C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 °C in the narrow temperature interval (3-4 °C). 4. The "acid state" is not accompanied by S-like transition at 25-35 °C. The thermal denaturation of the "acid state" occurs at 54 °C in the wide temperature interval (8-9 °C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 °C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.
INTRODUCTION We reported previously [1, 2] the existence of a reversible conformational transition of IgG fractions with different isoelectric points at pH 7.3-7.4 and the temperatures between 27 ± 2 and 38 :k 2 °C. The temperature dependent changes disappeared at pH values lower than 6.0 and higher than 8.5. A transition occurring at a high temperature was accompanied by a decrease in levorotation in the visible
156 region of the spectrum, intensification of the Cotton-effect minimum at the 228-225 nm region and an increase in the negative value of the parameter of the Moffit equation b0. A similar transition was observed for the myeloma IgG. The thermal perturbation difference spectra investigations showed an alteration in the asymmetric environment of tyrosine residues within the transition interval. These data and the early data [3-5] on lgG from human, bovine and horse blood serum showed that these changes occurring in physiological conditions are a very general IgG molecular property. In this paper we investigated the temperature and pH functions of the myeloma IgG (K) conformation by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and by difference adiabatic scanning microcalorimetry. The thermal difference spectra alterations as a result of antigen-antibody reaction were studied. MATERIALS AND METHODS
Purification of myeloma IgG. The blood serum was obtained from a hospital patient diagnosed as having multiple myeloma and the protein was precipitated with (NH4)zSO4 in the ratio 17.5 g of salt to 100 ml of serum. Purification was achieved by ion-exchange column chromatography on DEAE-cellulose (Whatman, England) using a gradient of increasing ionic strength (0.005-0.0175 M sodium phosphate) and decreasing pH (8.0-6.3). The absorbance of the fractions was measured at 280 nm. The major component was eluted with 0.01 M sodium phosphate buffer, pH 7.5. Further purification was achieved by gel filtration on Sephadex G-200. Typing. The myeloma immunoglobulin obtained from chromatography was typed by immunodiffusion in agar as G (K), using commercially prepared antisera to IgG, IgA, IgM, K- and L-chains (Behringwerke, G.F.R.). Homogeneity. The myeloma IgG (K) was submitted to electrofocusing in a carrier Ampholine using the Valmet horizontal apparatus of our own modification [6, 7]. Two fractions were obtained with isoelectric points of 6.15 and 6.5. The major component had an isoelectric point of 6.15. Specifically purified anti-DNS* antibody. Specifically purified rabbit anti-DNS antibody was isolated individually from the same immune serum using the same procedure as described previously [8]. Optical rotatory dispersion and circular diehroism. The optical rotatory dispersion over the wavelength range from 300 to 210 nm was investigated in a Jasco ORD/UV-5 spectrophotopolarimeter in a i mm long controlled-temperature cell. with protein concentrations from 0.05 to 0.07 ~ . CD over the wavelength range from 260 to 205 nm was investigated in a Jasco ORD/CD-20 spectropolarimeter in a 2 mm long controlled-temperature cell, with protein concentrations from 0.04 to 0.05~. The ORD over the wavelength range from 600 to 300 nm was investigated in a polarimeter with a two channel system of accumulation [9]. The iodine lamp was used as the source of light. The monochromatic light was obtained using the monochromator in combination with the interference light filters. The optical path was * DNS, l-dimethylaminonaphtalene-5-sulfonyl.
157 10 cm and the protein concentration was 0.4--0.8 ~. Purification with 0.22 nm millipore filters made it possible to obtain high-transparency solutions and reduce the error in the rotation-angle determinations to ± 0.0005 ° for the protein concentrations in question. Thermal perturbation difference spectra. The spectra were investigated in a Hitachi-124 spectrophotometer in 10 mm long controlled-temperature cells with protein concentrations from 0.04 to 0.07 ~ , Part of the spectra was investigated in a Specord UV VIS (Carl Zeiss, G.D.R.) spectrophotometer in 10-mm long controlledtemperature cells with protein concentration 0.106 ~ . Solvent perturbation difference spectra. These spectra were investigated in a Specord UV VIS spectrophotometer in a 10 mm long controlled-temperature tandem cells with protein concentration 0.106~. The perturbants employed in this study were all of spectroscopic grade. Difference adiabatic scanning microcalorimetry. These measurements were made with a DASM-1 [10] microcalorimeter in the thermodinamic laboratory of the Institute of Protein Research (Poustchino). The protein concentration was from 0.1 to 0.3~o. Electrochemical iodination. Selective modification of tyrosine residues was performed with the potentiostatic method of electrochemical iodination [11] in the electrochemical cell with the addition of sodium iodine. The protein concentration was 6.5 mg/ml, E = 545 mV, the volume was 6 ml, the time of the iodination was 20 min. The inorganic forms of iodine were removed using adsorbents described previously [12] and the percentage of combined iodine was determined [12]. The amount of mono-, and diiodtyrosine was determined by difference spectra between non-iodine and iodine protein at pH 13 [13]. pH measurements. Measurements of pH were made on a Radiometer pH meter (Denmark) and pH meter-246 (U.S.S.R.) with a glass electrode. Protein concentration, l~rotein concentration was determined from the absorbance at 280 nm with a Hitachi-124 and a Specord UV VIS spectrophotometer and a factor of 14.1 for a 1 g/100 ml of solution of IgG in 1.0 cm optical path. RESULTS
Exposure of lgG (K) chromophores at 20 °C The solvent perturbation difference spectra of IgG (K) in 20 ~o glycerol and in 2 0 ~ ethylene glycol are presented in Fig. 1A and lB. The curves were obtained at 20 °C in 0.01 M sodium phosphate buffer, pH 7.35. In order to make a quantitative estimate of the number of tyrosine and tryptophan residues exposed to perturbant, Okulov [14] has proposed the following equations: A E293(protein) = aTrp " A e~a
,d ezs7(protein) = aTrp • A E287 ra 0.4 +
(1) bTy r "
A e2M870.6
(2)
where a and b represent the number of tryptophans and tyrosines exposed. The Ae (protein) and Ae M values refer to molar absorption differences for the protein and
158 -0.01
260
280
300
320
3 4 0 ;k i'~m
-0.02
°t
0.02
,
287
i
260
i
2,;o
%f-J ~93-294
h
C ,
--
3~o 7 , ~
Fig. 1. The solvent perturbation difference spectra (A, B) and the thermal perturbation difference spectra (C) of IgG(K). The optical path was 1.0 cm and the protein concentration was 0.106 % (A, B) and 0.071 ~ (C). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The perturbants were 20~ glycerol (A) and 20~ ethylene glycol (B). The solvent perturbation difference spectra were measured at 20 °C. The measurements of the thermal perturbation difference spectra was made at the following conditions: The control cell temperature was 13.2 °C, the sample 23.8 °C.
the model compounds (56 Tyr and 22 Trp)[14] at the designated wavelengths. Typical results are presented in Table I. Fig. IC shows the thermal perturbation difference spectra of lgG (K) in 0.01 M phosphate buffer, pH 7.35. The control cell temperature was 13.2 °C and that of the sample was 23.8 °C. It can be seen that the spectra have two a b s o r b a n c e m a x i m a at 294 a n d 287 nm. It is k n o w n [15, 16] that the model spectra of tyrosine have a m a x i m u m at 287 n m a n d the model spectra of t r y p t o p h a n have a m i n i m u m at 292-293 nm. The longer wavelengths obtained for these m a x i m a of IgG (K) may
TABLE I EXPOSURE OF IMMUNOGLOBULIN G(K) CHROMOPHORES AT 20 °C Data calculated from the analysis of IgG(K) and the model compounds (56 tyrosine and 22 tryptophan) [14] in each perturbant according to equations (1) and (2) of text. Perturbant
de293" 10 s
,de~3" 103
No. of
/le287" 103
/1e2~7' 103
No. of
(20~)
(protein)
(model compounds)
exposed Trp
(protein)
(model compounds)
exposed Tyr
Ethylene glycol Glycerol
1.05 0.75
7.2 6.9
3 2
1.97 1.51
8.0 7.5
17 14
159 be connected with the definite contribution of the chromophores in the medium of low polarity. As the model system for IgG (K) we used a tyrosine solution in 40% ethylene glycol with a maximum spectra at 288 nm and a tryptophan solution in 60% ethylene glycol with a maximum spectra at 295 nm. In order to make a quantitative estimate of exposed chromophores Demchenko and Zyma [16] proposed the use of the extinction increment: (3) where 1 = 285-288 nm or 293-295 nm. The extinction increment is not dependent upon the temperature interval and the absolute absorption for Ezso 5 3. The extinction increment is dependent upon the polarity of the chromophore environment. The calculation of the number of tyrosine and tryptophan residues exposed to solvent was made using the following equations : o/oexposed Tyr = -
% exposed
Trp = -
Xii,” TYI x288
Xii,”
(5)
TIP
X295 at the temperature
intervals
IO-27 and 38-50 “C. The results are presented
in Table II.
TABLE II EXPOSURE t “C
OF IMMUNOGLOBULIN
Chromophores
G(K) CHROMOPHORES Chromophores
Protein I max
&so
A& -.104
X1.103
Ethylene glycol
0.782 0.524 0.193 0.427
40 60 40 60
At IO-27 38-50
Tyr Trp Tyr Trp
Temperature
287 295 287 295
dependent
0.368 0.632 0.368 0.632
2.9 3.3 0.71 2.7
A,,,,.
__ x;hr. lo3
% Exposed chromophores
No. of exposed chromophores
287 295 287 295
2.1 0.97 2.1 0.97
0.373 0.54 0.092 0.44
19.4 12 4.8 9.7
(%I
changes of IgG (K)
structure
Fig. 2A shows the optical rotation dependence on temperature at 551 nm. The solvent was 0.01 M sodium phosphate buffer, pHb7.35. The measurements were made on the same day of the protein preparation and 1 day and 5 days after preparation. The same protein solution was used for these investigations. Between the experiments the solution was kept in the spectropolarimetric cell at 4 “C. It can be seen that between 25 and 35 “C there were S-like transitions which were accompanied by an increase in the optical rotation. The changes outside the transition interval were observed. After storage the temperature dependence outside the transition range increases and the coefficient of the temperature dependence below the transition interval is equal to the coefficient of the temperature dependence above the transition
160
\
• 1 o 2 x3
O'tO 2b t,E
20
I,~o I I
40
t*~"
t *C
Fig. 2. The temperature function of the optical rotation (A) and the thermal perturbation difference spectra (B). The optical path was 10.0 cm (A), 1.0 cm (B). The protein concentration was 0.394 % (A) and 0.071% (B). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The measurements of the optical rotation (A) were made on the same day of protein preparation (1) and l day (2) and 5 days (3) after preparation. The thermal perturbation difference spectra were investigated at the following conditions: (B) The control cell temperature was 10 °C, the temperature of the sample was changed; (1) 287-288 nm, (2) 294-295 nm.
interval. The temperature dependence of the optical rotation outside the transition interval disappears when the concentration of the sodium phosphate buffer was increased to 0.1 M, but the parameters of the transition investigated are not altered. Fig. 3A shows the O R D curves for IgG (K) at pH 7.5 over the wavelength region from 300 to 210 nm at 20 and 40 °C. It can be seen that at 20 °C the O R D curve has a minimum at 224--225 nm and a shoulder at 228-229 nm. At 40 °C there was only a minimum at 228-229 nm. Fig. 3B shows the thermal perturbation difference spectra for IgG (K). The curves were obtained under the following conditions: (1) The control cell temperature 13.2, the sample 23.9 °C; (2) The control cell temperature 37.2, the sample 47.9 °C; (3) The control cell temperature 13.2, the sample 47.5 °C. The solvent was 0.01 M phosphate buffer, pH 6.9. It can be seen that the spectra below 25 °C and above 35 °C are significantly different, especially at 287 nm. Fig. 2B shows the temperature dependence of A E at 287 and 295 nm. It can be seen that the temperature dependence has an S-like form especially at 287 nm. The observed interval of the S-like transition coincides with the abrupt transition observed with optical rotation. The calculation of the exposed chromophores below and above the temperature transition is presented in Table II. It can be seen that the exposure of tyrosine residues decreases as a result of the temperature transition. We tested this effect by measuring the solvent perturbation difference spectra at 20 and at 40 °C using 20 % ethylene glycol as perturbant. The results of such a treatment are presented in Fig. 3C. It can be seen that the maximum at 287 nm decreases as a result of heating from 20 to 40 °C. We tested the effect on tyrosine residues screening by the electrochemical
161
[a(];~.l-3 O C 225 245 265 ;~nm -1 ........
i
3
-0,01 o
o.o~
B
AE'a~°
'
a~o
'
35o ' 3~o ~ "
- 0,011 0.011
~
=
,
260
C ,
28o
,
p
300
,
=
320 ;~ nm
Fig. 3. The temperature function of the optical rotation dispersion (A), the thermal perturbation difference spectra (B) and the solvent perturbation difference spectra (C) of IgG(K). The optical path was 0.1 cm (A) and 1.0 cm (B, C). The protein concentration was 0.071 ~ (B, (2) and 0.081 ~ (A). The solvent was 0.01 M sodium phosphate buffer, pH 7.35. The ORD (A) was measured at 20 °C (1) and at 40 °C (2). The thermal perturbation difference spectra were measured at the following conditions: The control cell temperature was 10 °C (1, 3) and 39 °C (2), the sample 20 °C (1) and 49 °C (2, 3). The solvent perturbation difference spectra (C) were measured at 20 °C (1) and at 40 °C (2). The perturbant was 20 ~ ethylene glycol. i o d i n a t i o n m e a s u r e m e n t s at 20 and 40 °C. T h e results are presented in Table III. It can be seen that at 40 °C the percentage o f c o m b i n e d iodine (at o t h er c o n s t a n t conditions) decreases by twice the value c o m p a r e d with the i o d i n a t i o n at 20 °C. Th e results o f i o d i n a t i o n in the m ix tu r e 1:4 are n o t in c o n t r a d i c t i o n with this conclusion because the a m o u n t o f m o n o i o d i n e tyrosines decreases by 6. We controlled the conf o r m a t i o n changes after the iodination. In a c c o r d a n c e with the C D data the conf o r m a t i o n o f I g G (K) is n o t altered u n d e r these conditions. TABLE III THE RESULTS OF THE ELECTROCHEMICAL IODINATION OF IgG(K) AT 20 AND 40 °C Protein/iodine mixture
20 °C
40 °C
Percentage of combined iodine
Amount of monoiodine Tyr (- 10s ,u/I)
Amount of diiodine Tyr (. l0 s ,u/I)
1 :I
85
--
_
1:2
99
--
_
1:4
83
10.85
3.01
1:8
79
--
_
Percentage of combined iodine
35
48 85 48
Amount of monoiodine Tyr (-105/~/l) --
-1.89 --
Amount of diiodine Tyr (" 10s/~/1) --
-6.72 --
162 We expected a heat effect as a result of the transition investigated, but the microcalorimetry measurements detected only a regular increase of the thermal heat capacity between the temperatures 20 and 50 “C. The pH-dependence
of the temperature
changes of IgG (K)
Fig. 4A shows the optical rotation temperature dependence of IgG (K) at different pH values. Fig. 4B shows the dependence of the temperature transition amplitude on pH. It can be seen that the temperature transition exists only in a narrow interval of pH: from 6.0 to 7.8. The transition amplitude reaches the maximum value at pH 6.9-7.0.
Fig. 4. The pH-function of the temperature changes of IgG(K) using the optical rotation (A, B) and the thermal perturbation difference spectra (C, D). The optical path was 10.0 cm (A, B) and 1.Ocm (C, D) and the protein concentration was 0.394% (A, B) and 0.13 % (C, D). The solvent was 0.01 M sodium phosphate buffer or 0.1 M acetate buffer at correspondent pH values. The measurements of the optical rotation (A) were made at the following pH values: 6.05 (l), 6.1 (2), 6.45 (3), 6.85 (4), 7.35 (5), 7.43 (6), 7.50 (7), 7.75 (8). (B) The pH-function of the amplitude of temperature transition of human myeloma IgG(K) (1) and the isoelectric fractions of IgG from bovine serum with p17.35 (2) and pZ7.95 (3). The pH-function of the thermal perturbation difference spectra (C, D) was measured at the following conditions: The control cell temperature was 23 “C, the sample 45 “C. (C, curve 1). The thermal perturbation difference spectra above pH 6.5. (C, curve 2) The spectra below pH 6.0. (D, curve x) E at 287-288 nm. (D, curve o) E at 294-295 nm.
Fig. 4C shows the temperature perturbation difference spectra of IgG (K) at different pH values. The temperature of the control cell was 23 “C, the sample temperature was 45 “C. The pH-dependence of the absorptivity difference at 287 nm and 295 nm is presented in Fig. 4D. It can be seen that the S-like transition occurs in the narrow interval of pH values (6.5-6.0). The S-like transition occurs in the narrow interval of pH values (6.5-6.0). The S-like transition occurring at lower pH is accom-
163 panied by an increase in the amplitude of the maxima at 287 and 295 nm and by a decrease in the optical rotation transition amplitude. It can be seen (Fig. 5) that the pH-dependence of a "high temperature state" (above 35 °C) is more than the pHdependence of a "low temperature state" (below 25 °C). At the physiological conditions of pH and temperature the exposure of tyrosine and tryptophan residues reaches the minimal value. -
tXE
O
.
O
1
~
(3
o.o1~-
a
v
m-
-
Fig, 5. The pH-function of the thermal perturbation difference spectra. The optical path was 1.0 cm. The protein concentration was 0.071 ~ . The solvent was 0.01 M sodium phosphate buffer or 0.] M
acetate buffer at correspondent pH values. The control cell temperature was 13.5 °C (1, 2) and 37 °C (3, 4), the sample 24.5 °C (1, 2) and 47 °C (3, 4); (1, 3), pH 7.0; (2, 4), pH 5.85. We investigated the thermal stability of the pH-dependent structural states. The results are presented in Fig. 6. It can be seen that the "acid state" does not accomplish the cooperative transition at 25-35 °C. The denaturation of the "acid state" occurs at 54 °C in the wide temperature interval (8-9 °C). After the thermal denaturation Trp residues are fully exposed to solvent and approx. 60 ~ of tyrosine residues are exposed. The "alkaline state" was found to be capable of fully reversible S-like transition at the temperatures between 25 and 35 °C (see Fig. 2B). The heating of IgG (K) from 45 to 63-64 °C is accompanied by the decrease of maxima at 287 and 295 nm. This process is linear and has a sign opposite to that for the thermal denaturation. The denaturation of the "alkaline state" occurs only at 64 °C in the narrow temperature interval (3-4 °C). The denaturation is accompanied by irreversible aggregation. According to the CD data there are no differences between the conformation for "acid" and "alkaline" states.
The thermal perturbation difference spectra of rabbit anti-DNS antibody before and after hapten-antibody reaction Fig. 7 shows the spectra of specifically purified rabbit anti-DNS antibody before and after hapten, antibody reaction. The temperature of the control cell was 17 °C, the sample 37 °C. The solvent was 0.005 M phosphate buffer with 0.15 M NaCI, pH 7.35. The hapten was added in a ratio of 2:1 to the protein. It can be seen that after hapten.antibody reaction the amplitude of the spectra sharply decreases especially at 287 and 295 nm. According to this result about 7 5 ~ of tyrosine and tryptophan residues are buried after hapten-antibody reaction. DISCUSSION According to the results obtained at pH 7.35 and 20 °C approx. 14 tyrosine and 2 tryptophan appear to be accessible to the glycerol; 17 tyrosine residues and
164
o0,I o
,.~
2~o~..
3oo
.~°~=
a / ~-" / / ~ J///7
o.o1~
~.~D"'.,;:/
/ ~ AE 1
ill
/
-oo~
,z_g
I,,,~ ~jr //,4"x
.,q\~ ~,~
/
0.01
"
i ~ ~E~ B
/x" •I
0.02
×2
//..7 /
o.oi
/ ×
/
~I J
x'
40
2b
oo
' t °c "~
Fig. 6. The thermal perturbation difference spectra of IgG(K). The optical path was 1.0 cm and the protein concentration was 0.047 7o. The solvent was 0.01 M sodium phosphate buffer, pH 7.0 (B and C, curve 1) and 0.1 M acetate buffer, pH 4.35 (A and C, curve 2). The temperature of a control cell was 13.6 °C. The temperature of the sample was changed in the following sequence: (A) 19.0 (1), 25 (2), 30.5 (3), 35.3 (4), 39.5 (5), 44.5 (6), 49.3 (7), 54.8 (8), 58.5 (9), 63.1 (10), 67 (11), 71.5 °C (12); (B) 45.5 (l), 48.5 (2), 51.5 (3), 54.5 (4), 57.5 (5), 60.6 (6), 63.6 (7), 66.6 °C (8). zXE
-
0.O1
oi
~10
~
~
2~0
2~0
'
~,~0 ~'~.
0.01 Fig. 7. The changes of the thermal perturbation difference spectra of the rabbit anti-DNS antibodies upon interaction with hapten. The optical path was 1.0 cm and the protein concentration was 0.054 ~ . The solvent was 0.005 M sodium phosphate buffer with 0.15 M NaCl. (1) The spectrum of free antibodies; (2) The spectrum of antibodies uoon interaction with hapten in molar ratio 1:2.
165 3 tryptophan residues are affected by the ethylene glycol and 19-20 tyrosine and 12 tryptophan are exposed according to the thermal perturbation difference spectra. The IgG (K) studied was found to be capable of a fully reversible S-like transition at p H values between 6.5 and 6.0. The changes occurring at lower p H are accompanied by an increase in exposure of the chromophores to solvent. In agreement with the results of the thermal denaturation the cooperativeness and stability of the "acid" state decreases sharply in comparison with the "alkaline state". This effect may be explained by the intramolecular dissociation of the domains due to ionisation of the imidazoles. The pH-changes investigated look very much like the pH-dependent dissociation of the light chain dimer [17]. The "alkaline state" was found to be capable of a fully reversible S-like transition at the temperatures between 25 and 35 °C. The changes occurring at the higher temperatures are accompanied by the screening of 14-15 tyrosine, but the transition is not accompanied by an appreciable heat effect. One of explanations of this contradiction may be the simulation of pseudo cooperativeness by the optical methods used. Another explanation may be the intramolecular compensation of the heat effect. A diagram of the states of IgG (K) is presented in Fig. 8. It can be seen that IgG (K) has some different states in conditions far from denaturation. :
. - _ ' . . ,
-,_."
.
.
. -._-
..'.'."
t *C
60
4O
20 I
i
i
i
i
2
4
6
8
10
pH
Fig. 8. The diagram of states of IgG(K) in the investigated region of pH and temperature values. The shade regions correspond to transition intervals. B, unfolded state; A~, folded state with a maximal screening of chromophores (associated); A~a, folded associated state above 45-50 °C; A~, folded "alkaline" state with a maximal exposure of chromophores (dissociated); A~, folded 'l'acid" state with a maximal exposure of chromophores. N, the region of existence of the native IgG structure at the physiological conditions of warm-blooded animals. Fig. 9 shows the general conformation of lgG (K) Fab fragment. The model is extrapolated as far as structural information is concerned partly from a Fab fragment [18, 19], and partly from a lambda chain dimer [20]. The numbering of amino acid residues in the K-chains is in accordance with alignment No. 50 [21], in the VH domain, in accordance with alignment No. 54 [21] and in the Cm domain, in accordance with alignment No. 55 [21 ]. Aromatic and charged side groups are marked using one letter code or the sign of the charge. The marked side chains are invariant in the K-chains observed. It can be seen that the invariant aromatic side chains are situated in the cavities between the domains and may be good markers for the investigation of the domain interposition. The ionisation of the invariant histidine
166 (+)1S
"rs
~
2
s8
6a
"~
VL
Fig. 9. The general conformation of ]gG(K) Fab fragment. The model is extrapolated as far as structural information is concerned, partly from a Fab fragment [18, ]9], and partly from a lambda chain dimer [20]. The numbering of amino acid residues in K-chains is in accordance with aligment No. 50 [21] and in Cm domain; in accordance with alignment No. 55 [2]], in VH domain; in accordance with alignment No. 54 [21 ]. Aromatic and charged side groups are marked using one letter code or the sign of the charge. The marked side chains are invarJant in K-chains observed.
residues situated in the cavity between the constant and variable domains may cause the pH transition studied. It is very important that a similar effect of about 75 chromophores screening is observed as a result of hapten, antibody interaction. This effect may be explained in the following way. At the temperature of warm-blooded animals (37-38 °C) all the molecules of antibody are in a "high temperature" native state. Only this state has a maximal affinity to the antigen. Thus the antigen stabilizes the "high temperature" native state of antibody. It is interesting that Pilz et al. [22] observed a decrease of the anti-poly(Dalanyl) antibodies volume by 1 0 ~ upon interaction with tetra-D-alanyl hapten. K/iiv/~r~iinen et al. [23] showed that the antigen, antibody interaction is accompanied by a decrease of the mobility of conjugated with histidine residues spin-labels. These investigations are in agreement with our results. The defect of the investigation of myeloma proteins with hapten-binding activity is the selection, in most cases, of specifically binding hapten at room temperatures. Thus one can explain the absence of structural alterations of myeloma proteins upon interaction with hapten [19, 24, 25] because the "low temperature" native state, in this case, is in the state of higher affinity. ACKNOWLEDGEMENTS The authors wish to thank Dr V. N. Kulakov of the Institute of Biophysics (Moscow) for assistance with the electrochemical iodination, Dr Chechinashvili, N.N. of the Institute of Protein Research (Poustchino) for assistance with the microcalorimetry measurements, Dr R. S. Nezlin of the Institute of Molecular Biology
167 (Moscow) for supply of rabbit anti-DNS serum and Dr V. I. Burlev of the Central Institute of Haematology and Blood Transfusion (Moscow) for supply of myeloma serum. The Authors thank Dr P. L. Privalov of the Institute of Protein Research (Poustchino) for helpful discussion of these results. REFERENCES 1 2 3 4 5 6 7 8 9 10
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