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Immunochemistry of sperm-whale myoglobin
Biochimica et Biophysica dcta, 328 (1973) 278-288 © Elsevier Scientific 19ublishing Company, Amsterdam - Printed in The Netherlands
BBA 36586 IMMUNOCHEMISTRY OF SPERM-WHALE MYOGLOBIN XVII. CONFORMATION AND IMMUNOCHEMISTRY OF DERIVATIVES MODIFIED AT LYSINES 98, 14o AND i45 BY REACTION W I T H 3,3-TETRAMETHYLENEGLUTARIC A N H Y D R I D E
M. Z. ATASSI, M. T. P E R L S T E I N AND D. J. STAUB
Department of Chemistry, Wayne State University, Detroit, Michigan (U.S.A.) (Received June 25th, 1973)
SUMMARY
Acylation of myoglobin (Mb) in the presence of IO molar excess of TGA gave a heterogeneous reaction product which showed four major components by gel electrophoresis. Chromatography on CM-cellulose yielded four electrophoreticallyhomogeneous tetramethyleneglutaryl-Mb components (TG-MbI, TG-MblI, TGMblII, TG-MblV). The number of lysine residues acylated agreed well with the increase in electrophoretic mobility. TG-MblV migrated like MbX and corresponded to residual unmodified MbX. The locations of the modified lysine residues in the derivatives were: TG-MbI, lysines 98, 14o and 145; TG-MblI, lysines 98 and 14o; TG-MblII, lysine 98. Absorption spectra of the four components were quantitatively identical with those of MbX. No conformational changes existed in TG-MbII or TG-MblII as determined from measurement of molecular parameters and from ORD and CD studies. Slight conformational changes were observed in TG-MbI. Immunochemical studies with antisera to MbX showed that, with a given antiserum, TGMbI, TG-MblI and TG-MblII had equal antigenic reactivity which was 15-2o% lower than the reaction of MbX with that antiserum. It was, therefore, concluded that lysine 98 is an antigenic reactive region in Mb. On the other hand, lysines 14o and 145 were clearly not part of a reactive region in Mb. From these findings and previously published results, it was possible to narrow down further a previously located antigenic reactive region in Mb so that it will now fall within sequence I46-151.
Abbreviations: ApoMb, apomyoglobin; Mb, metmyoglobin; MbX, the major chromatographic No. io obtained by CM-cellulose chromatographyS; TGA, 3,3-tetramethyleneglutaric anhydride; TG-Mb, 3,3-tetramethyleneglutalyl myoglobin; TG-MbI, TG-MbII, TG-MbIII, and TG-MbIV, refer to the corresponding chromatographic components of TG-Mb.
A N T I G E N I C S T R U C T U R E OF M Y O G L O B I N
279
INTRODUCTION
Delineation of the antigenic structure of native proteins is a task that demands a multifrontal attack for appropriate solution 1. Reactivity of a large number of peptides with varying overlaps must be studied, together with specific chemical derivatives of immunochemically reactive peptides. However, due to conformational and other factors1, 2, reliance on the fragmentation approach alone will unavoidably lead to erroneous conclusions. Independent evidence is obtained by studying the immunochemistry of highly purified and well characterized chemical derivatives of the intact native protein and also of closely related proteins with known structure and of peptides thereof. Clearly, studies of these proteins are meaningful only if supported by careful monitoring of conformational changes 3-6. Careful application of the aforementioned approaches should narrow down antigenic reactive regions to within a few residues of their actual size. Final delineation is achieved by synthesis and immunochemical studies of various parts of regions accurately narrowed down by the foregoing approaches. If synthesis precedes the orderly and careful chemical narrowing down, it will clearly be wasteful and in fact might lead to erroneous conclusions. Also, it is relevant, in this regard, to caution against isolating a set or two (say tryptic, chymotryptic) of peptides, studying their immunochemistry and then gaining the impression that the matter is solved. The complexity of the problem is such that none of the above approaches or even any combinations thereof can possibly yield the correct antigenic structure. The present communication describes the final stages in narrowing down the reactive region at the tail end of the Mb molecule (i.e. sequence 12o-153). Also, the results give further independent evidence for the presence of a reactive region around lysine 98. Three Mb derivatives modified at different lysine residues were prepared by reaction with 3,3-tetramethyleneglutaric anhydride 7. The conformation and immunochemistry of the derivatives are studied in detail. MATERIALS AND METHODS
Materials Metmyoglobin used here was the major chromatographic component No. io (MbX) obtained by CM-cellulose chromatography of crystalline Mb (ref. 8). This preparation was homogeneous by starch gel, acrylamide gel or disc electrophoresis. The preparation of ApoMb from MbX has already been described 9. The TGA was purchased from Aldrich Chemical Company. Reaction of MbX with TGA and chromatography of the reaction product To a magnetically stirred solution (4° m l ) of MbX (400 rag; 22.5 #moles) at o °C was added TGA (38. 4 rag; 228.5 #moles) in I i ml of acetone-water (I :2, v/v). Reaction mixture was stirred at o °C for 5 h after which it was dialyzed against eight changes ( 4 1 each) of water at o °C followed by dialysis against five changes of o.oi M NaH2PO 4 containing o.o1% KCN (pH 5.8). The reaction product was then centrifuged (4000 rev./min, o °C, 30 min) and applied onto a column (2.5 cm × 60 cm) of CM-cellulose which had been preequilibrated with o.oi M NaH2PO 4 containing o.o1% KCN (pH 5.8). The column was subjected to a linear pH gradient from pH 5.8
280
~i. Z. ATASSI el al.
to7.5.Themixingvesselcontainedo.olMNaH2PO, containingo.olO//o KCN, ~ . . pH 5.8 (I 1) and the reservoir contained o.oi M phosphate buffer, containing o.oi % KCN, p H 7.5 (2 1). Chromatography was at o °C and the column was eluted at the rate of 3 ° nil per h. Fractions (15 ml each) were read at 280, 420 and 54 ° n m . Fractions belonging to one peak were combined, concentrated in a Diaflo ultrafiltration cell (Amicon Corp., Cambridge, Mass.) at o °C, using N 2 (80 lb/inch 2) and a Diaflo membrane with a molecular weight exclusion limit of IOOO (Diaflo, UM-2 membrane). They were then examined by starch gel electrophoresis and peaks that showed heterogeneity (usually Peaks I and II) were subjected to rechromatography on the same column yielding completely homogeneous derivatives. The electrophoretically homogeneous components were finally dialyzed extensively against distilled water and then freeze dried.
A ntisera The preparation of rabbit and goat antisera to MbX has been described 1°. Antisera used in the present studies were goat antisera G3 and G4 and rabbit antiserum 77. Antisera were studied separately and were stored in 5-8-ml portions in the frozen state at --40 °C.
A ualytical methods Immunochemical methods (i.e. agar double diffusion, precipitin and absorption experiments) were performed as previously described 2. Absorbance measurements were done with a Zeiss PMQII spectrophotometer and continuous spectra were obtained on a Cary model 14 spectrophotometer. Procedure for starch gel electrophoresis has been described elsewhere n. Amino acid analyses of acid (double distilled, constant boiling HC1, IiO °C, 22 or 72 h, in N2-flushed evacuated sealed tubes) or alkaline 12 hydrolysates were carried out on a Spinco Model 12o C and/or BioCal BC-2oo amino acid analyzer. Tryptic digestion of ApoMb and ApoMb derivatives and their peptide mapping were done as described elsewhere 2. Peptide maps were stained with ninhydrin (0.2% in ethanol) or with specific stains for various amino acids la. Determination of the N-terminal (and also of the unreacted amino groups) was by amino acid analysis of acid hydrolysates of the derivatives after reaction with fluorodinitrobenzene as described elsewhere 1~. Concentrations of protein solutions were obtained from their N contents. N determinations were done with a micro-Kjeldahl procedure 15 and by using Nessler's reagent standardized with (NH4)2SO 4. At least, triplicate analyses were routinely carried out and they varied ± 0.5% or less. The N2 contents of MbX and the present three derivatives (calculated from their amino acid compositions) are: MbX, 17.36%; TG-MbI, 16.88%" TG-MbII, i7.o4% ' TG-MbIII, 17.2o°/,.
Evaluation of conformational changes Determination of Stokes radius (a) and frictional ratios (fifo) was done by gel filtration on calibrated Sephadex G-75 columns as described previously in detail4, n. Conformational changes were also monitored by ORD and CD measurements of the protein solutions in water. The ORD and CD results are given here in Ern'j~ and iO'J~, respectively. Experimental procedure and quantitative treatment of data have been described in detail elsewhere1", a4. The molecular weight and mean residue weight values used here are: MbX, 17 816 and 116.4; TG-MbI, 18 320 and 119.74; TG-
ANTIGENIC STRUCTURE OF MYOGLOBIN
281
MbII, z8 z52 and II8.64; T G - M b I I I , 17 984 and II7.54, respectively. For the calculation of bo (ref. 17) , a ~o value of 216 nm was employed. RESULTS
Reaction of MbX with TGA and purification of the derivatives A previous report 7 has shown that reaction of MbX with 55 molar excess of TGA resulted in the modification of 16 out of 19 lysine residues. Therefore, in the present work, conditions for the preparation of less drastically modified derivatives were investigated. Small scale reactions were carried out on portions (5 rag) of MbX in saturated sodium acetate (0. 5 ml) with 3, 6, IO, 20 and 55 molar excess of TGA. Reactions were carried out at o °C for 5 h, after which they were examined by starch gel electrophoresis. Reaction of MbX in 55 molar excess of TGA gave a single very negatively charged band with a mobility 6.91 (relative to MbX = I.OO). Reaction with 20 molar excess gave this band of highly modified derivative (mobility 6.90 ) and a less negatively charged band (mobility 5.02) in approximately equal amounts. Reaction with IO molar excess of TGA gave four electrophoretic components in approximately even distribution of mobilities 5.07; 3.97; 2-50; and i.oo. Reactions with 3 and 6 molar excess of TGA showed a large amount of unreacted MbX and only one other component with mobility of 2.50. From these studies, it was clear that only a small fraction of the total protein was modified on reaction with 3 and 6 molar excess of TGA. On the other extreme, reactions with 20 and 55 molar excess of TGA gave derivatives that were too extensively modified. Reaction in IO molar excess, on the other hand, gave derivatives that m a y have possessed a gradation in the extent of modification. I t was, therefore, decided to carry out the large scale reactions of MbX with IO molar excess of TGA at o°C for 5 h .
1.4 i.2
1.0 .~0.8 0.6
0.l 0.2
5LO I(~0 150 FRACTION NUMBER Fig. I. C h r o m a t o g r a p h i c p a t t e r n of T G - M b (4oo mg) on a CM-cellulose c o l u m n (2.5 c m × 60 cm). I s - m l f r a c t i o n s were collected. F o r details, see t e x t .
282
M.z. ATASSI et al.
In Fig. I the chromatographic pattern of the TG-Mb large scale reaction product is shown. It can be seen that four chromatographic components were obtained. Component I and, in some preparations, Component I I needed rechromatography. Starch gel electrophoresis (Fig. 2) of TG-Mb chromatographic components showed that these were highly homogeneous. In gel electrophoresis, TG-MbIV superimposed with MbX.
Fig. 2. Starch gel electrophoresis of TG-Mb and its chromatographic components. I, TG-MbI; II, TG-MblI; III, TG-MblII; IV, TG-MblV; V, TG-Mb (i.e. reaction product of MbX with TGA). Electrophoretic mobility of TG-MblV superimposed with that of MbX.
Determination of the modified residues Amino acid analysis of the chromatographic components TG-MbI, TG-MbII, T G - M b l I I and TG-MblV showed that the lysine contents of the derivatives decreased to varying extents (Table I). The number of lysine residues modified were (in moles/ mole Mb): TG-MbI, 3; TG-MblI, 2; TG-MblII, i. No modification was detected in TG-MblV suggesting that this component corresponded to some unreacted native Mb left in the reaction mixture. The decrease in the lysine content was accompanied by the appearance of a new peak; corresponding to that of 3,3-tetramethyleneglutarimidolysine which appears on the analyzer 33 min after phenylalanine 7. Accurate determination of 3,3-tetramethyleneglutarimidolysine, was not successful due to the broad shape of the peak and the low number of lysine residues modified. Extent of modification was further confirmed by reaction with fluorodinitrobenzene. After reaction with fluorodinitrobenzene, acid hydrolysates (72 h) revealed that the following number of lysine residues did not react with fluorodinitrobenzene : TG-MbI, 2.6; TG-MblI, 1.8; T G - M b l I I , I . I ; TG-MblV, o, confirming that these were modified by reaction with TGA. Also, the valine content in each of the D N P derivatives
283
ANTIGENIC STRUCTURE OF MYOGLOBIN TABLE I AMINO ACID COMPOSITION OF MbX AND DERIVATIVES
The amino acid compositions are given ill residues per mole. The results represent the average of four acid hydrolyses (two 22- and two 72-h hydrolyses). Values for serine and threonine were obtained b y extrapolation to zero hydrolysis time. T r y p t o p h a n was determined from alkaline hydrolysis. N.D., not determined. A m i n o acid
MbX
TG-MbI
TG-MblI
TG-MblII
TG-MbI V
Trp Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe
1.93 19.1o 12.oo 4.16 7.92 4-79 5.87 18.78 4.15 11.o8 16.87 7 .82 2.02 8.58 18.27 2.95 6.11
2.04 16.35 12.oo 3.85 8.04 4.84 5.68 18.74 4.14 11.38 17.2o 7.67 1.95 8.50 17.72 2.88 5.82
1.86 17.1o 11.61 4.05 8-07 5 .00 5.72 19.o 3 4.09 11.o 3 16.98 7.72 1.89 8.60 17.6o 2.83 5.89
N.D. 18.o4 12.oo 3.92 7.99 5.13 6.12 19.26 4.00 11.o8 17.o6 7.83 1.74 8.43 17.88 2.83 5.84
1.82 18.97 11.9o 4.03 8.29 4.75 5.86 18.61 4.00 lO.97 16.7o 7 .80 1.79 8.95 18.27 3.28 6.08
decreased by one residue indicating that the N-terminal valine had not reacted with TGA. Locations of the modified residues were determined by peptide mapping of tryptic hydrolysates of ApoMb preparations derived from each of the TG-Mb chromatographic components. Fig. 3 shows a peptide map outline for ApoMb, on which also is indicated the spots that disappear in each of TG-MbI, TG-MblI and TGMbIII. Identity of the spots shown in the map in Fig. 3, relative to the known amino acid sequence of Mb (ref. 18), was determined by elution, after cutting out, from several lightly stained maps (0.05% ninhydrin in ethanol), centrifugation, freeze drying, acid hydrolysis and then amino acid analysis. New spots appeared in the derivatives and due to their overlap with other peptides their compositions were not determined. The results are summarized in Fig. 3- In TG-MblII, peptides 97-98 and 99-1o2 disappeared indicating that lysine 98 was modified. With TG-MblI, peptides 97-98, 99-1o2 and 141-145 disappeared completely indicating that lysine 98 was also modified in this derivative together with lysine 14o. These two lysine residues (i.e. 98 and 14o) were again modified in TG-MbI together with lysine 145 since, in addition, peptide 146-147 disappeared in this derivative.
Eleetrophoretic and spectral properties of the derivatives Each of the present TG-Mb components was electrophoretically homogeneous (Fig. 2) and was more negatively charged than MbX. The electrophoretic mobility increased linearly with increase in the nmnber of lysine residues modified (at least up to three modified lysine residues). Mobilities of the derivatives (relative to MbX = I.OO) were: TG-MbI, 5.06; TG-MblI, 3.85; TG-Mb-III, 2.48 and TG-MblV, I.OO. Spectral studies were carried out on the cyanmet forms of MbX and of the TG-Mb
.~. Z. ATASSIet al.
284
6 A
® C, ©
0 O
Fig. 3. A tracing of the tryptic peptide map of ApoMb. By elution, hydrolysis and amino acid analysis and comparison with the known sequence of Mb TM, spots in the map had the following sequences: (i) 148-153; (2) 141-145; (3) 35-42; (4) 43-45; (5) 51-56; (6) 57-62; (7) 119-133; (8) 80-87; (9) 79-87; (io) 88-96; (II) 97-98; (12) 78-79; (13) 17-31; (14) 48-50; (15) 146 147; (16) 99-1o2; (17) 46-47; (18) 97-1o2, appears in the three TG-Mb derivatives; (19) 134-139; (20) 32-34 . In TG-MblII, peptides i i and 16 disappeared completely and a new Spot A corresponding to sequence 97-1o2 appeared. In TG-MblI, peptides i i and 16 again disappeared and, ill addition, peptide 2 disappeared completely. In TG-MbI, peptides i i, 16, 2 and 15 disappeared completely. The aforementioned peptides t h a t disappear in various derivatives are marked by an arrow. The new spots that appear in various derivatives are shaded. Spot A appears in TG-MblII, Spots A and 13 appear in TG-MbII, and Spots A and C appear in TG-MbI.
derivatives. The results indicated that MbX and all the TG-Mb components had identical spectral properties. Each protein gave absorption maxima at 279-280, 359-360, 422-423, and 54 ° n m . The molar extinction at each maximum was unaltered in the derivative relative to MbX.
Evaluation of conformational changes Values for Stokes radius (a) and forf/fo in TG-MblI, TG-MbIII and MbX were identical (Table II). On the other hand, these two molecular parameters were slightly, but significantly increased in TG-MbI. It is relevant to mention here that the change observed with TG-MbI was well outside the present experimental variation which was -i- 0.7% or less. In ORD studies the present three TG-Mb derivatives, like MbX, all gave a negative rotation minimum at 233 nm and a positive extremum at 199 nm. The two derivatives, TG-MblI and TG-MblII, had equal rotatory power to that of MbX both at the negative minimum and at the positive maximum. Also, the b0 values were comparable. On the other hand, TG-MbI was less rotatory at these two bands and also its b0 was appreciably lower. Table II summarizes the ORD parameters for these proteins. In CD measurements the present proteins showed negative ellipticity bands at 220 and 208 nm. The CD spectra for TG-MbI, TG-MblI, TG-MblII and for MbX were determined in the range 260 to 200 nm. The ellipticity bands for MbX, TG-MblI and TG-MblII were equal in magnitude, both at 208 and 220 nm.
285
ANTIGENIC STRUCTURE OF MYOGLOBIN TABLE II CONFORMATIONAL PARAMETERS OF M b X AND DERIVATIVES
Gel f i l t r a t i o n v a l u e s were o b t a i n e d f r o m t h r e e r e p l i c a t e d e t e r m i n a t i o n s a n d v a r i e d 4-o.7 % o r l e s s ,
TG-MbI TG-MblI TG-MbIII TG-MbIV MbX
Hydrodynamic parameters (from gel filtration)
ORD parameters*
a (A)
f/fo
[m']23s
[m']19,
bo
[0"]2,0
[0".72o8
20. 5 19.o 19.1 19.o 19.o
1.17 1.o9 1.o9 1.o9 1.o9
--8 --8 --9 --9 --9
+4 ° +46 +43 +46 +46
--299 --424 --435 --420 --417
--17 --19 -- 19 --19 --19
--18 --21 --zo --2o --20
ioo 82o 45o 35o 32o
CD parameters*
520 46o 92o ioo 360
920 630 300 400 5o0
750 350 840 600 200
* M e a s u r e m e n t s were c a r r i e d o u t on t h e p r o t e i n s o l u t i o n s in w a t e r .
The EO'] values for these two bands were slightly decreased in TG-MbI. The CD parameters are also summarized in Table I I.
Immunochemistry of the derivatives In agar double diffusion with antisera to MbX, each of TG-MbI, TG-MblI and TG-MblII, gave a single sharp precipitin line which fused completely with the line given by MbX, showing no spurs or intersections. In quantitative precipitin analysis, the present three derivatives showed lower antigenic reactivity than that of MbX. With a given antiserum, the reactivities of TG-MbI, TG-MblI and TG-MblII were equal and, for the present three sera, ranged between 80-85 % relative to the reaction
001 0.4
E
o
0.2
0
t
| I 2.0 4.0 6.0 Antigen nitrogen (pg)
Fig. 4. Q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n s of M b X (O), T G - M b I (O ), T G - M b l I (A), T G - M b l I I ( I ) , a n d T G - M b l V ([]) w i t h a n t i s e r u m G 4. F o r assay, p r e c i p i t a t e s w e re d i s s o l v e d in 0. 5 m l of 0. 5 M N a O H a n d t h e n d e t e r m i n e d w i t h t h e F o l i n - L o w r y m e t h o d sa.
286
5~. z. ATASSI c'l al.
of MbX taken as IOOCl:'o. Fig. 4 shows an example of the precipitin reaction of the three TG-Mb derivatives and MbX with antiserum G 4. The antigenic reactivities of the three derivatives, relative to MbX, with all the present three antisera are summarized in Table I I I . Absorption experiments with G4 confirmed that each of the TG-Mb derivatives removed 8o-82~}~) of the serum reactivity toward MbX. Also, absorption with any of the derivatives completely removed the antiserum reactivity with all the others. Since TG-MbIV had no modified amino acids and corresponded to residual unreacted native protein which was subjected to all conditions of reaction and manipulations of preparation, it should represent a very appropriate control. TABLE III Q U A N T I T A T I V E P R E C I P I T I N R E A C T I O N S OF T G - M b
D E R I V A T I V E S V~ITH V A R I O U S A N T I S E R A TO M b X
Values are g i v e n in p e r c e n t p r e c i p i t a t i o n at e q u i v a l e n c e relative to r e a c t i o n of M b X w i t h t h e respective a n t i s e r u m . R e s u l t s r e p r e s e n t t h e a v e r a g e of f o u r or m o r e d e t e r m i n a t i o n s a n d varied ~ I . I ° ~ , or less. G3 a n d G 4 are two g o a t a n t i s e r a a n d 77 is a r a b b i t a n t i s e r u m , each to MbX.
Protein
TG-MbI TG-MblI TG-MbIII TG-MblV
% Reaclion with a~tiserum G3
G4
77
84.3 81.6 84. 5 99.6
81.7 81. 5 83. 4 98.7
79.4 81.9 79.9 98.5
With all three MbX antisera, TG-MbIV showed equal antigenic reactivity to that of MbX (Table III). Therefore, the observed changes in immunoehemical behaviors of TG-MbI, TG-MbII and T G - M b l I I were caused by the modification itself and not by the conditions of the reaction. DISCUSSION
Reaction of lysine residues in Mb with TGA was previously studied 7 under conditions that were intended to drive the reaction to completion. Therefore, the nature of the lysine reaction product and its stability to acid hydrolysis has already been reporte&. It is noteworthy that the lysine residues in Mb, although virtually all exposed 19, showed remarkable differences in their accessibility to reaction with TGA. This is not unusual, and differences in accessibility among the carboxyl groupsQ,2°; the arginines21; the tyrosines1°,22; the tryptophans n and the histidines ~a 2~ have been observed. Similar differences in accessibility were also observed for reaction of the lysine residues in lysozyme6, 27. Spectral measurements reflected that the heme environment of the three TG-Mb derivatives was unchanged, relative to MbX. A small conformational alteration, suggested from gel filtration of TG-MbI, was further confirmed b y the results of ORD and CD measurements. Clearly, no conformational changes take place upon modification of lysine 9 8 (TG-MbIII) or of lysine 98 and 14o (TG-MbII). However, when lysines 9 8 and 14o are modified together with lysine 145, the protein undergoes a certain amount of conformational change due, most likely, to the cooperative effect of modifying two lysines (i.e. 14o and 1 4 5 ) simultaneously by a bulky negatively charged substituent in one helix (i.e. helix H).
ANTIGENIC STRUCTURE OF MYOGLOBIN
287
It is significant that the immunochemical reactivity decreased by 15-2o °/o upon modification of lysine 98. In view of the absence of conformational changes, which could influence antigenic reactivity3,5, ~1, it may be concluded that the decrease in reactivity was directly caused by the modification of lysine 98. The introduction of a negatively charged bulky substituent at the lysyl side chain would be expected to eliminate the reaction of the whole antigenic reactive region. For example, succinylation of lysine 16 in the reactive region 16 21 (present in sequence 1-55 of Mb), obliterates the immunochemical reaction of that region entirely ~s. A reactive region has been narrowed down to fall within (but does not include all of) sequence 86-1o2 of Mb(refs 9, 20) and it accounts for 16-2o% of the total antigenic reactivity of the proteinZ, ~9. The present findings show unambiguously that lysine 98 forms an essential part of this region. No further decrease in antigenic reactivity was observed when both lysine 98 and 14o were modified simultaneously (TG-MblI). Since the derivative showed no conformational changes, it is not difficult to see that lysine 14 ° does not fall within an antigenic reactive region in Mb. The case of TG-MbI merits special discussion. This derivative, in which lysines 98, 14o, and 145 were modified, showed some conformational alterations. However, its immunochemical behavior resembled that of TG-MblI, and TG-MbIII. Since TGMblII has only lysine 98 modified and the same lysine residue is modified in the other two TG-Mb derivatives, it is clear that modification of lysines 14o and 145 make no further contribution to the decrease in antigenic reactivity. Also, the confor~ mational change in TG-MbI had no destructive effect on the antigenic reactivity. This was unexpected but not unusual since it is now well demonstrated that conformational changes will not always influence antigenic reactivity and the effect will rather depend on the protein and on the nature and extent of conformational change~0, 31. Previous studies from this laboratory (see following paper 32 for review of evidence) have shown that a single antigenic reactive region occurs in the entire sequence 12o-153 and is located within (but does not include all of) the sequence 14o-151. The present findings show that lysines 14o and 145 are not part of the reactive region and since the space interval between them is far too short to contain a complete antigenic reactive region, it must be concluded that the reactive region commences after lysine 145 and occupies the entire sequence 146-151, at the end of helix H and part of the random C-terminal pentapeptide. In the following communication 3~, from the immunochemistry of various synthetic parts of this region, it is confirmed that this reactive region occupies exclusively the sequence 146-151 . In conclusion, from reaction of MbX with TGA, three derivatives were prepared that were modified at lysine 98, at lysines 98 and 14o or at lysines 98, 14o and 145. Modification of lysine 98 alone or of lysine 98 and 14o together resulted in no change in conformation while modification of the three lysines 98, 14o and 145 induced some conformational alteration. Immunochemical studies confirmed the presence of a reactive region centered around lysine 98. Also, the results narrowed down a previously located antigenic reactive region so that it now falls within the sequence 146151. In the following paper, the chemical synthesis and immunochemistry of various parts of this region are given.
M.Z. ATASSI c t a l .
288 ACKNOWLEDGEMENTS
T h i s w o r k w a s s u p p o r t e d b y a g r a n t (AM 13389) f r o m t h e N a t i o n a l I n s t i t u t e o f A r t h r i t i s a n d M e t a b o l i c D i s e a s e s , N a t i o n a l I n s t i t u t e s o f H e a l t h , U.S. P u b l i c H e a l t h Service.
REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34
Atassi, M. Z. (1972) Third Int. Convoc. Immunol., in the press Atassi, M. Z. and Saplin, B. J. (1968) Biochemistry 7, 688-698 Atassi, M. Z. (1967) Biochem. J. lO3, 29-35 Atassi, M. Z. (197 o) Biochim. Biophys. Acta 221, 612-622 Andres, S. F. and Atassi, M. Z. (197 o) Biochemistry 9, 2268-2275 Habeeb, A. F. S. A. and Atassi, M. Z. (1971) Bioehim. Biophys. Acta 236, 131-141 Atassi, M. Z. (1967) Biochem. J. lO2, 488-491 Atassi, M. Z. (1964) Nature 202, 496-498 Atassi, M. Z. and Perlstein, M. T. (1972) Biochemistry II, 3984-399 ° Atassi, M. Z. (1967) Biochem. J. lO2, 478-487 Atassi, M. Z. and Caruso, D. R. (1968) Biochemistry 7, 699-7 °6 Ray, W. J. and Koshland, D. E. (1962) J. Biol. Chem. 237, 2493-2505 Easley, C. W. (1965) Biochim. Biophys. deta lO7, 386-388 Atassi, M. Z. and Saplin, B. J. (1966) Bioehem. J. 98, 82-93 Markham, R. (1942) Biochem. J. 79o-791 Singhal, R. P. and Atassi, M. Z. (197o) Biochemistry 9, 4252-4259 Moffitt, W. and Yang, J. T. (1956) Proc. Natl. Acad. Sci. U.S. 42, 596-603 Edmundson, A. B. (1965) Nature 205, 883-887 Kendrew, J. C., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, D. C. and Shore, V. C. (1961) Nature 19o, 666-670 Atassi, M. Z. and Singhal, R. P. (1972) Immunochemistry 9, lO57-1o66 Atassi, M. Z. and Thomas, A. V. (1969) Biochemistry 8, 3385-3394 Hermans, J. and Wan Lu, L. (1967) Arch. Biochem. Biophys, 122, 331-337 Banaszak, L. J., Andrews, P. A., Burger, J. W., Eylar, E. H. and Gurd, F. R. N. (1963) J. Biol Chem. 238, 33o7-3314 Hugli, T. E. and Gurd, F. R. N. (197° ) J. Biol. Chem. 245, 193o-1938 Hugli, T. E. and Gurd, F. R. N. (197o) J. Biol. Chem. 245, 1939-1946 Andres, S. F. (1972) Ph.D. Thesis, Wayne State University Habeeb, A. F. S. A. and Atassi, M. Z. (1971) Immunochemistry 8, lO47-1o59 Perlstein, M. T. and Atassi, M. Z. (1973) Immunochemistry, in the press Atassi, M. Z. and Singhal, R. P. (197 o) Biochemistry 9, 3854-3861 Atassi, M. Z. and Habeeb, A. F. S. A. (1969) Immunoehemistry 6, 555-566 Atassi, M. Z,, Perlstein, M. T. and Habeeb, A. F. S. A. (1971) J. Biol. Chem. 246, 3291 3296 Koketsu, J. and Atassi, M. Z. (1973) Biochim. Biophys. Acta 328, 289-302 Lowry, O. H., Rosebrough, N. J. and Randall, R. J. (1951) J. Biol. Chem. I93, 265-275 Atassi, M. Z. and Singhal, R. P. (1972) J. Biol. Chem. 247, 5980-5986
BBA 36586 IMMUNOCHEMISTRY OF SPERM-WHALE MYOGLOBIN XVII. CONFORMATION AND IMMUNOCHEMISTRY OF DERIVATIVES MODIFIED AT LYSINES 98, 14o AND i45 BY REACTION W I T H 3,3-TETRAMETHYLENEGLUTARIC A N H Y D R I D E
M. Z. ATASSI, M. T. P E R L S T E I N AND D. J. STAUB
Department of Chemistry, Wayne State University, Detroit, Michigan (U.S.A.) (Received June 25th, 1973)
SUMMARY
Acylation of myoglobin (Mb) in the presence of IO molar excess of TGA gave a heterogeneous reaction product which showed four major components by gel electrophoresis. Chromatography on CM-cellulose yielded four electrophoreticallyhomogeneous tetramethyleneglutaryl-Mb components (TG-MbI, TG-MblI, TGMblII, TG-MblV). The number of lysine residues acylated agreed well with the increase in electrophoretic mobility. TG-MblV migrated like MbX and corresponded to residual unmodified MbX. The locations of the modified lysine residues in the derivatives were: TG-MbI, lysines 98, 14o and 145; TG-MblI, lysines 98 and 14o; TG-MblII, lysine 98. Absorption spectra of the four components were quantitatively identical with those of MbX. No conformational changes existed in TG-MbII or TG-MblII as determined from measurement of molecular parameters and from ORD and CD studies. Slight conformational changes were observed in TG-MbI. Immunochemical studies with antisera to MbX showed that, with a given antiserum, TGMbI, TG-MblI and TG-MblII had equal antigenic reactivity which was 15-2o% lower than the reaction of MbX with that antiserum. It was, therefore, concluded that lysine 98 is an antigenic reactive region in Mb. On the other hand, lysines 14o and 145 were clearly not part of a reactive region in Mb. From these findings and previously published results, it was possible to narrow down further a previously located antigenic reactive region in Mb so that it will now fall within sequence I46-151.
Abbreviations: ApoMb, apomyoglobin; Mb, metmyoglobin; MbX, the major chromatographic No. io obtained by CM-cellulose chromatographyS; TGA, 3,3-tetramethyleneglutaric anhydride; TG-Mb, 3,3-tetramethyleneglutalyl myoglobin; TG-MbI, TG-MbII, TG-MbIII, and TG-MbIV, refer to the corresponding chromatographic components of TG-Mb.
A N T I G E N I C S T R U C T U R E OF M Y O G L O B I N
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INTRODUCTION
Delineation of the antigenic structure of native proteins is a task that demands a multifrontal attack for appropriate solution 1. Reactivity of a large number of peptides with varying overlaps must be studied, together with specific chemical derivatives of immunochemically reactive peptides. However, due to conformational and other factors1, 2, reliance on the fragmentation approach alone will unavoidably lead to erroneous conclusions. Independent evidence is obtained by studying the immunochemistry of highly purified and well characterized chemical derivatives of the intact native protein and also of closely related proteins with known structure and of peptides thereof. Clearly, studies of these proteins are meaningful only if supported by careful monitoring of conformational changes 3-6. Careful application of the aforementioned approaches should narrow down antigenic reactive regions to within a few residues of their actual size. Final delineation is achieved by synthesis and immunochemical studies of various parts of regions accurately narrowed down by the foregoing approaches. If synthesis precedes the orderly and careful chemical narrowing down, it will clearly be wasteful and in fact might lead to erroneous conclusions. Also, it is relevant, in this regard, to caution against isolating a set or two (say tryptic, chymotryptic) of peptides, studying their immunochemistry and then gaining the impression that the matter is solved. The complexity of the problem is such that none of the above approaches or even any combinations thereof can possibly yield the correct antigenic structure. The present communication describes the final stages in narrowing down the reactive region at the tail end of the Mb molecule (i.e. sequence 12o-153). Also, the results give further independent evidence for the presence of a reactive region around lysine 98. Three Mb derivatives modified at different lysine residues were prepared by reaction with 3,3-tetramethyleneglutaric anhydride 7. The conformation and immunochemistry of the derivatives are studied in detail. MATERIALS AND METHODS
Materials Metmyoglobin used here was the major chromatographic component No. io (MbX) obtained by CM-cellulose chromatography of crystalline Mb (ref. 8). This preparation was homogeneous by starch gel, acrylamide gel or disc electrophoresis. The preparation of ApoMb from MbX has already been described 9. The TGA was purchased from Aldrich Chemical Company. Reaction of MbX with TGA and chromatography of the reaction product To a magnetically stirred solution (4° m l ) of MbX (400 rag; 22.5 #moles) at o °C was added TGA (38. 4 rag; 228.5 #moles) in I i ml of acetone-water (I :2, v/v). Reaction mixture was stirred at o °C for 5 h after which it was dialyzed against eight changes ( 4 1 each) of water at o °C followed by dialysis against five changes of o.oi M NaH2PO 4 containing o.o1% KCN (pH 5.8). The reaction product was then centrifuged (4000 rev./min, o °C, 30 min) and applied onto a column (2.5 cm × 60 cm) of CM-cellulose which had been preequilibrated with o.oi M NaH2PO 4 containing o.o1% KCN (pH 5.8). The column was subjected to a linear pH gradient from pH 5.8
280
~i. Z. ATASSI el al.
to7.5.Themixingvesselcontainedo.olMNaH2PO, containingo.olO//o KCN, ~ . . pH 5.8 (I 1) and the reservoir contained o.oi M phosphate buffer, containing o.oi % KCN, p H 7.5 (2 1). Chromatography was at o °C and the column was eluted at the rate of 3 ° nil per h. Fractions (15 ml each) were read at 280, 420 and 54 ° n m . Fractions belonging to one peak were combined, concentrated in a Diaflo ultrafiltration cell (Amicon Corp., Cambridge, Mass.) at o °C, using N 2 (80 lb/inch 2) and a Diaflo membrane with a molecular weight exclusion limit of IOOO (Diaflo, UM-2 membrane). They were then examined by starch gel electrophoresis and peaks that showed heterogeneity (usually Peaks I and II) were subjected to rechromatography on the same column yielding completely homogeneous derivatives. The electrophoretically homogeneous components were finally dialyzed extensively against distilled water and then freeze dried.
A ntisera The preparation of rabbit and goat antisera to MbX has been described 1°. Antisera used in the present studies were goat antisera G3 and G4 and rabbit antiserum 77. Antisera were studied separately and were stored in 5-8-ml portions in the frozen state at --40 °C.
A ualytical methods Immunochemical methods (i.e. agar double diffusion, precipitin and absorption experiments) were performed as previously described 2. Absorbance measurements were done with a Zeiss PMQII spectrophotometer and continuous spectra were obtained on a Cary model 14 spectrophotometer. Procedure for starch gel electrophoresis has been described elsewhere n. Amino acid analyses of acid (double distilled, constant boiling HC1, IiO °C, 22 or 72 h, in N2-flushed evacuated sealed tubes) or alkaline 12 hydrolysates were carried out on a Spinco Model 12o C and/or BioCal BC-2oo amino acid analyzer. Tryptic digestion of ApoMb and ApoMb derivatives and their peptide mapping were done as described elsewhere 2. Peptide maps were stained with ninhydrin (0.2% in ethanol) or with specific stains for various amino acids la. Determination of the N-terminal (and also of the unreacted amino groups) was by amino acid analysis of acid hydrolysates of the derivatives after reaction with fluorodinitrobenzene as described elsewhere 1~. Concentrations of protein solutions were obtained from their N contents. N determinations were done with a micro-Kjeldahl procedure 15 and by using Nessler's reagent standardized with (NH4)2SO 4. At least, triplicate analyses were routinely carried out and they varied ± 0.5% or less. The N2 contents of MbX and the present three derivatives (calculated from their amino acid compositions) are: MbX, 17.36%; TG-MbI, 16.88%" TG-MbII, i7.o4% ' TG-MbIII, 17.2o°/,.
Evaluation of conformational changes Determination of Stokes radius (a) and frictional ratios (fifo) was done by gel filtration on calibrated Sephadex G-75 columns as described previously in detail4, n. Conformational changes were also monitored by ORD and CD measurements of the protein solutions in water. The ORD and CD results are given here in Ern'j~ and iO'J~, respectively. Experimental procedure and quantitative treatment of data have been described in detail elsewhere1", a4. The molecular weight and mean residue weight values used here are: MbX, 17 816 and 116.4; TG-MbI, 18 320 and 119.74; TG-
ANTIGENIC STRUCTURE OF MYOGLOBIN
281
MbII, z8 z52 and II8.64; T G - M b I I I , 17 984 and II7.54, respectively. For the calculation of bo (ref. 17) , a ~o value of 216 nm was employed. RESULTS
Reaction of MbX with TGA and purification of the derivatives A previous report 7 has shown that reaction of MbX with 55 molar excess of TGA resulted in the modification of 16 out of 19 lysine residues. Therefore, in the present work, conditions for the preparation of less drastically modified derivatives were investigated. Small scale reactions were carried out on portions (5 rag) of MbX in saturated sodium acetate (0. 5 ml) with 3, 6, IO, 20 and 55 molar excess of TGA. Reactions were carried out at o °C for 5 h, after which they were examined by starch gel electrophoresis. Reaction of MbX in 55 molar excess of TGA gave a single very negatively charged band with a mobility 6.91 (relative to MbX = I.OO). Reaction with 20 molar excess gave this band of highly modified derivative (mobility 6.90 ) and a less negatively charged band (mobility 5.02) in approximately equal amounts. Reaction with IO molar excess of TGA gave four electrophoretic components in approximately even distribution of mobilities 5.07; 3.97; 2-50; and i.oo. Reactions with 3 and 6 molar excess of TGA showed a large amount of unreacted MbX and only one other component with mobility of 2.50. From these studies, it was clear that only a small fraction of the total protein was modified on reaction with 3 and 6 molar excess of TGA. On the other extreme, reactions with 20 and 55 molar excess of TGA gave derivatives that were too extensively modified. Reaction in IO molar excess, on the other hand, gave derivatives that m a y have possessed a gradation in the extent of modification. I t was, therefore, decided to carry out the large scale reactions of MbX with IO molar excess of TGA at o°C for 5 h .
1.4 i.2
1.0 .~0.8 0.6
0.l 0.2
5LO I(~0 150 FRACTION NUMBER Fig. I. C h r o m a t o g r a p h i c p a t t e r n of T G - M b (4oo mg) on a CM-cellulose c o l u m n (2.5 c m × 60 cm). I s - m l f r a c t i o n s were collected. F o r details, see t e x t .
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M.z. ATASSI et al.
In Fig. I the chromatographic pattern of the TG-Mb large scale reaction product is shown. It can be seen that four chromatographic components were obtained. Component I and, in some preparations, Component I I needed rechromatography. Starch gel electrophoresis (Fig. 2) of TG-Mb chromatographic components showed that these were highly homogeneous. In gel electrophoresis, TG-MbIV superimposed with MbX.
Fig. 2. Starch gel electrophoresis of TG-Mb and its chromatographic components. I, TG-MbI; II, TG-MblI; III, TG-MblII; IV, TG-MblV; V, TG-Mb (i.e. reaction product of MbX with TGA). Electrophoretic mobility of TG-MblV superimposed with that of MbX.
Determination of the modified residues Amino acid analysis of the chromatographic components TG-MbI, TG-MbII, T G - M b l I I and TG-MblV showed that the lysine contents of the derivatives decreased to varying extents (Table I). The number of lysine residues modified were (in moles/ mole Mb): TG-MbI, 3; TG-MblI, 2; TG-MblII, i. No modification was detected in TG-MblV suggesting that this component corresponded to some unreacted native Mb left in the reaction mixture. The decrease in the lysine content was accompanied by the appearance of a new peak; corresponding to that of 3,3-tetramethyleneglutarimidolysine which appears on the analyzer 33 min after phenylalanine 7. Accurate determination of 3,3-tetramethyleneglutarimidolysine, was not successful due to the broad shape of the peak and the low number of lysine residues modified. Extent of modification was further confirmed by reaction with fluorodinitrobenzene. After reaction with fluorodinitrobenzene, acid hydrolysates (72 h) revealed that the following number of lysine residues did not react with fluorodinitrobenzene : TG-MbI, 2.6; TG-MblI, 1.8; T G - M b l I I , I . I ; TG-MblV, o, confirming that these were modified by reaction with TGA. Also, the valine content in each of the D N P derivatives
283
ANTIGENIC STRUCTURE OF MYOGLOBIN TABLE I AMINO ACID COMPOSITION OF MbX AND DERIVATIVES
The amino acid compositions are given ill residues per mole. The results represent the average of four acid hydrolyses (two 22- and two 72-h hydrolyses). Values for serine and threonine were obtained b y extrapolation to zero hydrolysis time. T r y p t o p h a n was determined from alkaline hydrolysis. N.D., not determined. A m i n o acid
MbX
TG-MbI
TG-MblI
TG-MblII
TG-MbI V
Trp Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe
1.93 19.1o 12.oo 4.16 7.92 4-79 5.87 18.78 4.15 11.o8 16.87 7 .82 2.02 8.58 18.27 2.95 6.11
2.04 16.35 12.oo 3.85 8.04 4.84 5.68 18.74 4.14 11.38 17.2o 7.67 1.95 8.50 17.72 2.88 5.82
1.86 17.1o 11.61 4.05 8-07 5 .00 5.72 19.o 3 4.09 11.o 3 16.98 7.72 1.89 8.60 17.6o 2.83 5.89
N.D. 18.o4 12.oo 3.92 7.99 5.13 6.12 19.26 4.00 11.o8 17.o6 7.83 1.74 8.43 17.88 2.83 5.84
1.82 18.97 11.9o 4.03 8.29 4.75 5.86 18.61 4.00 lO.97 16.7o 7 .80 1.79 8.95 18.27 3.28 6.08
decreased by one residue indicating that the N-terminal valine had not reacted with TGA. Locations of the modified residues were determined by peptide mapping of tryptic hydrolysates of ApoMb preparations derived from each of the TG-Mb chromatographic components. Fig. 3 shows a peptide map outline for ApoMb, on which also is indicated the spots that disappear in each of TG-MbI, TG-MblI and TGMbIII. Identity of the spots shown in the map in Fig. 3, relative to the known amino acid sequence of Mb (ref. 18), was determined by elution, after cutting out, from several lightly stained maps (0.05% ninhydrin in ethanol), centrifugation, freeze drying, acid hydrolysis and then amino acid analysis. New spots appeared in the derivatives and due to their overlap with other peptides their compositions were not determined. The results are summarized in Fig. 3- In TG-MblII, peptides 97-98 and 99-1o2 disappeared indicating that lysine 98 was modified. With TG-MblI, peptides 97-98, 99-1o2 and 141-145 disappeared completely indicating that lysine 98 was also modified in this derivative together with lysine 14o. These two lysine residues (i.e. 98 and 14o) were again modified in TG-MbI together with lysine 145 since, in addition, peptide 146-147 disappeared in this derivative.
Eleetrophoretic and spectral properties of the derivatives Each of the present TG-Mb components was electrophoretically homogeneous (Fig. 2) and was more negatively charged than MbX. The electrophoretic mobility increased linearly with increase in the nmnber of lysine residues modified (at least up to three modified lysine residues). Mobilities of the derivatives (relative to MbX = I.OO) were: TG-MbI, 5.06; TG-MblI, 3.85; TG-Mb-III, 2.48 and TG-MblV, I.OO. Spectral studies were carried out on the cyanmet forms of MbX and of the TG-Mb
.~. Z. ATASSIet al.
284
6 A
® C, ©
0 O
Fig. 3. A tracing of the tryptic peptide map of ApoMb. By elution, hydrolysis and amino acid analysis and comparison with the known sequence of Mb TM, spots in the map had the following sequences: (i) 148-153; (2) 141-145; (3) 35-42; (4) 43-45; (5) 51-56; (6) 57-62; (7) 119-133; (8) 80-87; (9) 79-87; (io) 88-96; (II) 97-98; (12) 78-79; (13) 17-31; (14) 48-50; (15) 146 147; (16) 99-1o2; (17) 46-47; (18) 97-1o2, appears in the three TG-Mb derivatives; (19) 134-139; (20) 32-34 . In TG-MblII, peptides i i and 16 disappeared completely and a new Spot A corresponding to sequence 97-1o2 appeared. In TG-MblI, peptides i i and 16 again disappeared and, ill addition, peptide 2 disappeared completely. In TG-MbI, peptides i i, 16, 2 and 15 disappeared completely. The aforementioned peptides t h a t disappear in various derivatives are marked by an arrow. The new spots that appear in various derivatives are shaded. Spot A appears in TG-MblII, Spots A and 13 appear in TG-MbII, and Spots A and C appear in TG-MbI.
derivatives. The results indicated that MbX and all the TG-Mb components had identical spectral properties. Each protein gave absorption maxima at 279-280, 359-360, 422-423, and 54 ° n m . The molar extinction at each maximum was unaltered in the derivative relative to MbX.
Evaluation of conformational changes Values for Stokes radius (a) and forf/fo in TG-MblI, TG-MbIII and MbX were identical (Table II). On the other hand, these two molecular parameters were slightly, but significantly increased in TG-MbI. It is relevant to mention here that the change observed with TG-MbI was well outside the present experimental variation which was -i- 0.7% or less. In ORD studies the present three TG-Mb derivatives, like MbX, all gave a negative rotation minimum at 233 nm and a positive extremum at 199 nm. The two derivatives, TG-MblI and TG-MblII, had equal rotatory power to that of MbX both at the negative minimum and at the positive maximum. Also, the b0 values were comparable. On the other hand, TG-MbI was less rotatory at these two bands and also its b0 was appreciably lower. Table II summarizes the ORD parameters for these proteins. In CD measurements the present proteins showed negative ellipticity bands at 220 and 208 nm. The CD spectra for TG-MbI, TG-MblI, TG-MblII and for MbX were determined in the range 260 to 200 nm. The ellipticity bands for MbX, TG-MblI and TG-MblII were equal in magnitude, both at 208 and 220 nm.
285
ANTIGENIC STRUCTURE OF MYOGLOBIN TABLE II CONFORMATIONAL PARAMETERS OF M b X AND DERIVATIVES
Gel f i l t r a t i o n v a l u e s were o b t a i n e d f r o m t h r e e r e p l i c a t e d e t e r m i n a t i o n s a n d v a r i e d 4-o.7 % o r l e s s ,
TG-MbI TG-MblI TG-MbIII TG-MbIV MbX
Hydrodynamic parameters (from gel filtration)
ORD parameters*
a (A)
f/fo
[m']23s
[m']19,
bo
[0"]2,0
[0".72o8
20. 5 19.o 19.1 19.o 19.o
1.17 1.o9 1.o9 1.o9 1.o9
--8 --8 --9 --9 --9
+4 ° +46 +43 +46 +46
--299 --424 --435 --420 --417
--17 --19 -- 19 --19 --19
--18 --21 --zo --2o --20
ioo 82o 45o 35o 32o
CD parameters*
520 46o 92o ioo 360
920 630 300 400 5o0
750 350 840 600 200
* M e a s u r e m e n t s were c a r r i e d o u t on t h e p r o t e i n s o l u t i o n s in w a t e r .
The EO'] values for these two bands were slightly decreased in TG-MbI. The CD parameters are also summarized in Table I I.
Immunochemistry of the derivatives In agar double diffusion with antisera to MbX, each of TG-MbI, TG-MblI and TG-MblII, gave a single sharp precipitin line which fused completely with the line given by MbX, showing no spurs or intersections. In quantitative precipitin analysis, the present three derivatives showed lower antigenic reactivity than that of MbX. With a given antiserum, the reactivities of TG-MbI, TG-MblI and TG-MblII were equal and, for the present three sera, ranged between 80-85 % relative to the reaction
001 0.4
E
o
0.2
0
t
| I 2.0 4.0 6.0 Antigen nitrogen (pg)
Fig. 4. Q u a n t i t a t i v e p r e c i p i t i n r e a c t i o n s of M b X (O), T G - M b I (O ), T G - M b l I (A), T G - M b l I I ( I ) , a n d T G - M b l V ([]) w i t h a n t i s e r u m G 4. F o r assay, p r e c i p i t a t e s w e re d i s s o l v e d in 0. 5 m l of 0. 5 M N a O H a n d t h e n d e t e r m i n e d w i t h t h e F o l i n - L o w r y m e t h o d sa.
286
5~. z. ATASSI c'l al.
of MbX taken as IOOCl:'o. Fig. 4 shows an example of the precipitin reaction of the three TG-Mb derivatives and MbX with antiserum G 4. The antigenic reactivities of the three derivatives, relative to MbX, with all the present three antisera are summarized in Table I I I . Absorption experiments with G4 confirmed that each of the TG-Mb derivatives removed 8o-82~}~) of the serum reactivity toward MbX. Also, absorption with any of the derivatives completely removed the antiserum reactivity with all the others. Since TG-MbIV had no modified amino acids and corresponded to residual unreacted native protein which was subjected to all conditions of reaction and manipulations of preparation, it should represent a very appropriate control. TABLE III Q U A N T I T A T I V E P R E C I P I T I N R E A C T I O N S OF T G - M b
D E R I V A T I V E S V~ITH V A R I O U S A N T I S E R A TO M b X
Values are g i v e n in p e r c e n t p r e c i p i t a t i o n at e q u i v a l e n c e relative to r e a c t i o n of M b X w i t h t h e respective a n t i s e r u m . R e s u l t s r e p r e s e n t t h e a v e r a g e of f o u r or m o r e d e t e r m i n a t i o n s a n d varied ~ I . I ° ~ , or less. G3 a n d G 4 are two g o a t a n t i s e r a a n d 77 is a r a b b i t a n t i s e r u m , each to MbX.
Protein
TG-MbI TG-MblI TG-MbIII TG-MblV
% Reaclion with a~tiserum G3
G4
77
84.3 81.6 84. 5 99.6
81.7 81. 5 83. 4 98.7
79.4 81.9 79.9 98.5
With all three MbX antisera, TG-MbIV showed equal antigenic reactivity to that of MbX (Table III). Therefore, the observed changes in immunoehemical behaviors of TG-MbI, TG-MbII and T G - M b l I I were caused by the modification itself and not by the conditions of the reaction. DISCUSSION
Reaction of lysine residues in Mb with TGA was previously studied 7 under conditions that were intended to drive the reaction to completion. Therefore, the nature of the lysine reaction product and its stability to acid hydrolysis has already been reporte&. It is noteworthy that the lysine residues in Mb, although virtually all exposed 19, showed remarkable differences in their accessibility to reaction with TGA. This is not unusual, and differences in accessibility among the carboxyl groupsQ,2°; the arginines21; the tyrosines1°,22; the tryptophans n and the histidines ~a 2~ have been observed. Similar differences in accessibility were also observed for reaction of the lysine residues in lysozyme6, 27. Spectral measurements reflected that the heme environment of the three TG-Mb derivatives was unchanged, relative to MbX. A small conformational alteration, suggested from gel filtration of TG-MbI, was further confirmed b y the results of ORD and CD measurements. Clearly, no conformational changes take place upon modification of lysine 9 8 (TG-MbIII) or of lysine 98 and 14o (TG-MbII). However, when lysines 9 8 and 14o are modified together with lysine 145, the protein undergoes a certain amount of conformational change due, most likely, to the cooperative effect of modifying two lysines (i.e. 14o and 1 4 5 ) simultaneously by a bulky negatively charged substituent in one helix (i.e. helix H).
ANTIGENIC STRUCTURE OF MYOGLOBIN
287
It is significant that the immunochemical reactivity decreased by 15-2o °/o upon modification of lysine 98. In view of the absence of conformational changes, which could influence antigenic reactivity3,5, ~1, it may be concluded that the decrease in reactivity was directly caused by the modification of lysine 98. The introduction of a negatively charged bulky substituent at the lysyl side chain would be expected to eliminate the reaction of the whole antigenic reactive region. For example, succinylation of lysine 16 in the reactive region 16 21 (present in sequence 1-55 of Mb), obliterates the immunochemical reaction of that region entirely ~s. A reactive region has been narrowed down to fall within (but does not include all of) sequence 86-1o2 of Mb(refs 9, 20) and it accounts for 16-2o% of the total antigenic reactivity of the proteinZ, ~9. The present findings show unambiguously that lysine 98 forms an essential part of this region. No further decrease in antigenic reactivity was observed when both lysine 98 and 14o were modified simultaneously (TG-MblI). Since the derivative showed no conformational changes, it is not difficult to see that lysine 14 ° does not fall within an antigenic reactive region in Mb. The case of TG-MbI merits special discussion. This derivative, in which lysines 98, 14o, and 145 were modified, showed some conformational alterations. However, its immunochemical behavior resembled that of TG-MblI, and TG-MbIII. Since TGMblII has only lysine 98 modified and the same lysine residue is modified in the other two TG-Mb derivatives, it is clear that modification of lysines 14o and 145 make no further contribution to the decrease in antigenic reactivity. Also, the confor~ mational change in TG-MbI had no destructive effect on the antigenic reactivity. This was unexpected but not unusual since it is now well demonstrated that conformational changes will not always influence antigenic reactivity and the effect will rather depend on the protein and on the nature and extent of conformational change~0, 31. Previous studies from this laboratory (see following paper 32 for review of evidence) have shown that a single antigenic reactive region occurs in the entire sequence 12o-153 and is located within (but does not include all of) the sequence 14o-151. The present findings show that lysines 14o and 145 are not part of the reactive region and since the space interval between them is far too short to contain a complete antigenic reactive region, it must be concluded that the reactive region commences after lysine 145 and occupies the entire sequence 146-151, at the end of helix H and part of the random C-terminal pentapeptide. In the following communication 3~, from the immunochemistry of various synthetic parts of this region, it is confirmed that this reactive region occupies exclusively the sequence 146-151 . In conclusion, from reaction of MbX with TGA, three derivatives were prepared that were modified at lysine 98, at lysines 98 and 14o or at lysines 98, 14o and 145. Modification of lysine 98 alone or of lysine 98 and 14o together resulted in no change in conformation while modification of the three lysines 98, 14o and 145 induced some conformational alteration. Immunochemical studies confirmed the presence of a reactive region centered around lysine 98. Also, the results narrowed down a previously located antigenic reactive region so that it now falls within the sequence 146151. In the following paper, the chemical synthesis and immunochemistry of various parts of this region are given.
M.Z. ATASSI c t a l .
288 ACKNOWLEDGEMENTS
T h i s w o r k w a s s u p p o r t e d b y a g r a n t (AM 13389) f r o m t h e N a t i o n a l I n s t i t u t e o f A r t h r i t i s a n d M e t a b o l i c D i s e a s e s , N a t i o n a l I n s t i t u t e s o f H e a l t h , U.S. P u b l i c H e a l t h Service.
REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34
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