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A differential method for determining the relative reactivity to iodination of different tyrosyl residues in a protein molecule
BIOCHIMICA ET BIOPHYSICA ACTA
I
BBA 3 5 1 1 2
A DIFFERENTIAL METHOD FOR DETERMINING THE RELATIVE R E A C T I V I T Y TO I O D I N A T I O N OF D I F F E R E N T T Y R O S Y L R E S I D U E S IN A P R O T E I N MOLECULE O. A. R O H O L T AND D. P R E S S M A N
Department of Biochemistry Research, Roswell Park Memorial Institute*, Buffalo, N.Y. (U.S.A.) (Received May 22nd, 1967)
SUMMARY
(I) A method has been developed for determining the relative rates of iodination of various tyrosyl residues in a protein. This is done b y the use of a paired-label technique in which one portion of the protein is iodinated with iodine labeled with 125I and another portion is iodinated to incorporate a different number of iodine atoms labeled with 131I. The proteins are mixed and enzymatically digested. The resulting iodinated peptides are separated. Each iodinated peptide separated is a mixture of the 131I-labeled peptide from the first iodination and the 125I-labeled peptide from the second iodination. (2) The relative reactivities of particular tyrosyl residues (identified by surrounding sequences) toward iodination can be determined from an analysis of the ratios of the amounts of iodine incorporated in the corresponding tyrosyl peptides derived from each of the two iodinated preparations. (3) There is no need to recover quantitatively all the monoiodotyrosine and diiodotyrosine peptides derived from a particular tyrosine, a recovery which is difficult since for a single tyrosyl residue both of these forms can be distributed among several peptides.
INTRODUCTION
All of the various tyrosyl residues in a protein molecule do not seem to be exposed to the same degree. Experiments involving the iodination of several antibodies have shown that the iodination of tyrosyl residues is largely responsible for a loss of antibody activity and that the tyrosyl in the combining site seems to be iodinated at a rate more rapid than the average rate for all tyrosyl residues l-a. In the case of ribonuclease, it is relatively easy to iodinate some of the tyrosyl residues while others are iodinated very slowly under the same conditions 4. Of the tyrosyl residues in insulin, those of the A chain are iodinated in the intact molecule more readily than those of the B chain according to SPRINGELL~ and DE ZOETEN AND DE BRUIN6. They have taken this to mean that different tyrosyl residues are exposed to different extents. In the case of ribonuclease and bovine serum albumin, the ab* A u n i t of the New Y o r k State D e p a r t m e n t of Health.
Biochim. Biophys. Acta, 147 (1967) 1-14
2
o . A . ROHOLT, D. PRESSMAN
sorption spectrum varies for the different tyrosyl residues and this also indicates different exposure 7. We have found that the reactivities of the four tyrosyl residues of native ~-chymotrypsin towards iodination are in the order 146 > 94 > 171 > 229 with relative reactivities of I : 0.4: 0.2: 0.02, respectivelys. We have studied the relative rates of iodination of different residues in a protein and have been able to isolate peptides containing tyrosyl residues of differing reactivities toward iodination. This was done by a paired-label technique9,1°, where one portion of the protein was iodinated to a certain level with iodine labeled with 125I and another portion was iodinated to a somewhat higher level of iodination with iodine containing la1I. These two iodinated preparations were then mixed and digested with pepsin and the resulting peptides were separated by high-voltage paper electrophoresis and chromatography. Differences in the amounts of each iodinated peptide as determined from the two iodine isotopes indicated the relative degrees of reactivity towards iodination of various tyrosyl residues in the protein. The protein studied was a portion of the antibody molecule which had been isolated following papain digestion of the antibody molecule and which was of particular interest since it contains the combining site".
MATERIALS AND METHODS
Antibody fraction A papain digest 11 of specifically purified rabbit anti-/5-azobenzoate antibody" was chromatographed on CM-cellulose and the fraction designated as I I c in our previous reporO 2 was used.
Enzymes Pronase (enzyme P from Streptomyces griseus) was obtained from Biddle Sawyer Company, New York, N.Y. and pancreatin from Merck and Company, Rahway, N.J.
Iodination of protein Iodinations were carried out as described previously using hypoiodite labeled with either 1251 or 1311 (see ref. 9)-
Isolation of iodinated ])eptides Mixtures of 1251- and 131I-labeled protein in 0.5 M HCOOH were digested with pepsin (protein-pepsin, 30: I, by weight) at 37 ° for 15-2o h. The digests were subjected to chromatography (n-butanol-acetic acid-water, 4:1:5, by vol.,) on W h a t m a n 3 MM paper followed by high-voltage paper electrophoresis (High Voltage Electrophoretor, Model D, Gilson Medical Electronics). The separations were carried out at 40 V per cm using 4 % HCOOH as the background electrolyte. Radioautographs of the dried paper were prepared b y exposing it to Kodak Industrial X - R a y Film Type KK1 for suitable periods. Spots on the radioautograph were sequentially numbered and the area on the paper corresponding to each spot was cut out and the 1251/131I ratio for each spot was calculated after counting in an automatic recording, dual-channel g a m m a - r a y spectrometer. Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
3
RESULTS
Variations in the amount of iodinated peptide obtained following peptic digestion of a protein iodinated to different levels Three 2.5-ml portions of a solution containing 4.1 mg of the protein were each exposed to a different concentration of hypoiodite labeled with 125I. The level of iodine incorporation was 6. 4 , 7.4 and 8. 4 iodine atoms per antibody fragment (50000, tool. wt.). A fourth portion was iodinated to the level of 8.1 iodine atoms per fragment with lslI-labeled iodine, and served as the reference protein against which the others were compared. A portion of each of the three l~SI-labeled proteins was individually mixed with an equal weight of the lS~I-labeled protein to give three paired mixtures. The paired mixtures were digested with pepsin. Portions of the digest were subjected to paper chromatography and to high-voltage paper electrophoresis and radioautographs were prepared. The patterns of spots on the radioautographs were similar since they were all due primarily to the ~31I label of the single preparation of reference protein above. Corresponding spots on each radioautograph were readily recognized*. All the spots ATOMS I
PER MOLECULE
8.4 (125T), 8.[ (131I) (0)
,:oE 1.5[
z
Z4 (125I), 8.t (131I)
H .J
1'5 f 1.0 0.8 0.6
I5
34 13
:r?o
'
L,
25
l''"
,b,
30
mH 2.0
14
r
I
IO
t
34
8 TTT
T .
64 (t25 I) I~
8"I (t311)
(c)
19
,T T,
l,
T
50
I 000
t
I
I0 000 13[I (COUNTS PER MINUTE PER SPOT )
I00 000
Fig, I. Relative a m o u n t s of iodinated peptides obtained from F r a c t i o n I I c of an anti-X~ a n t i b o d y following iodination to different levels. E a c h p o i n t represents the results for an individual spot cut from the electrophoretogram. The o r d i n a t e shows the ratio of the a m o u n t of 12SI-labeled iodine to the a m o u n t of 131I-labeled iodine, i.e., the ratio of the a m o u n t s of a given iodinated peptide derived from each of the t w o iodinated protein p r e p a r a t i o n s t h a t were mixed and digested. On the abscissa, the 1311 c o u n t per s p o t is shown. * E v e n t h o u g h the same a m o u n t of the same lSlI-labeled protein was t a k e n for (a), (b) or (c) of Fig. i, the c o u n t rate of 1811 observed for a given spot m a y n o t be the same due to variations in the area of the s p o t cut from the p a p e r ; this does n o t affect the value of the ratio for the spot, however, unless the s p o t is a poorly resolved mixture.
Biochim. Biophys. Acta, 147 (1967) 1-14
4
O . A . R O H O L T , D. PRESSMAN
were numbered for identification and comparison. Some of the nmnbers are given on Fig. I. The ratio of the amount of ~25I-labeled iodine to the amount of 131I-labeled iodine was then determined for each spot. This ratio is equivalent to the ratio of the amount of the iodinated peptide obtained from each of the paired proteins. Ratios for the three paired proteins are shown in Fig. I. The solid line represents the quotient of the number of iodine atoms per protein molecule at the low level of iodination and the number at the high level of iodination. In the case of the pair for which the level of iodination was nearly the same, 8.4 and 8.1 iodine atoms per molecule, the amount of each of the radioactive peptides obtained from each of the proteins was the same (Fig. Ia). In the other mixtures, where the differences in the levels of iodination were greater (Fig. ib, c), the ratios for m a n y of the iodinated peptides deviated rather widely from one another.
Determination of the presence of monoiodotyrosine and of diiodotyrosine in the isolated, iodinated peptides In another experiment, 5 mg of the antibody protein was iodinated with i25Ilabeled iodine and a second portion was iodinated with l~lI-labeled iodine but to a higher level. The level of iodine incorporation was found to be 6.6 4 iodine atoms per protein molecule for the 125I-labeled material and 7.63 for the ~3~I-labeled material. Equal weights of the two were mixed and digested with pepsin. I mg of the digested protein was subjected to chromatography followed by high-voltage paper electrophoresis and a radioautograph was prepared. The ratio of 125I-labeled iodine to ~31I-1abeled iodine was determined for each spot as shown in Figs. 2a, b. The ratios for selected spots are shown in Table I. The presence of monoiodotyrosine or of diiodotyrosine in the peptide associated with these selected spots was then determined. Twelve spots with ratios greater than 1.o5, including nine spots with the highest ratios (1.25-2.43) and three of the most TABLE
I
SPOTS SELECTED
FROM PATTERN
S H O W N O N P I G . 23, F O R A N A L Y S I S O F M O N O - A N D D I I O D O T Y R O S I N E
Analyzed individually Ratio ~ z.o5 Spot No.
Ratio
26 28 29 51 53 66 74 135 149 15o 151 152
1.97 2.13 1.87 1-47 2.43 1.66 1.5° 1.52 1.25 i.io 1.o 7 1.16
Pooled for analysis Ratio ~ 0.96
125I
(counts~rain) I 060 2 600 9 lo 5 2oo 13 3 ° 0 3 600 2 920 2 760 6 760 18 800 22 800 22 200
Spot No.
Ratio
32 54 55 56 57 69 83 86 87 89
0.64 0.82 o.81 0.82 0.86 0.76 0.72 0.92 0.96 0.96
Ratio I.o5-I.24 12~I
(counts~rain)
Biochim. Biophys. ,4cta, 147 (1967) I 14
i 24 19 io 9 8 2 17 19 19
250 400 ooo 300 020 090 OlO 300 700 200
Spot No.
Ratio
43 52 73 118 132 146 147 148 158 159 165 166 167
I.O9 1.24 1.o 9 1.17 1.09 1.16 I.IO 1.o 9 1.13 1.22 1.31
125I (counts/rain) 7 67o 45 ° I 12o 47 ° 4 800 2 960 6 ooo 12 600 3 34 ° 860 I 320
1.21
I 600
1.2o
I 480
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
5
radioactive spots (ratios 1.o7-1.16 ) were individually analyzed for monoiodotyrosine and diiodotyrosine. Additionally, ten spots with ratios less than o.96 were individually analyzed for monoiodotyrosine and diiodotyrosine. Thirteen other spots with ratios between 1.o5 and 1.24 were treated as a pool (see Table I).
ATOMS "r PER MOLECULE 6.6 ( } z s I ) , 7.6( 131"r ) H
3I
2O .2 H
10 08
,,~?,
,'
r!
~ -,r,.
'X~
0 o
101000
100 131I (COUNTS PER MINUTE PER SPOT)
Fig. 2a. R a d i o a u t o g r a p h of iodinated peptides separated from a peptic digest of iodinated antib o d y f r a g m e n t b y c h r o m a t o g r a p h y a n d high-voltage paper electrophoresis. Peptides eluted from the spots indicated b y arrows were analyzed for t h e presence of mono- or diiodotyrosine. The q u a n t i t a t i v e d a t a concerning each of t h e spots are in Table I. Fig. 2b. Same as Fig. I b u t for a n o t h e r preparation of antibody.
The analyses were carried out by pulping the paper in minimal volumes of i M NH4OH and the pulp filtered on a flitted funnel and then similarly extracted two more times. Over 95 % of the radioactivity was recovered in a volume of less than 2 ml. The eluates were lyophilized in Io-ml erlenmeyer flasks. Hydrolysis of the peptides was done b y adding 0. 4 ml of 0.05 M ammonium bicarbonate containing o.I mg of pronase to each flask and incubating at 37 ° for 4 h. The contents were lyophilized and a further digestion using pancreatin was then carried out in the same manner. The digests were again lyophilized and the residues taken up in minimal volumes of I M formic acid containing carrier monoiodotyrosine, diiodotyrosine, Biochirn. Biophys. Acta, 147 (I967) 1-14
6
O. A. ROHOLT, D. PRESSMAN
monoiodohistidine and diiodohistidine. This was applied to 1.25 inch Whatman 3 MM strips and subjected to high-voltage paper electrophoresis in I M formic acid in order to separate the iodinated compound. Radioautographs were then prepared. Areas on the paper which corresponded to the radioactivity were cut out and counted. The paper strips were reconstituted and the positions of all of the iodinated compounds located by means of a ceric sulfate-arsenious acid spray 13. Comparison of the sprayed strip and the radioautograph permitted an unambiguous identification of the radioactive material as monoiodotyrosine or as diiodotyrosine. Neither radioiodinated monoiodohistidine nor diiodohistidine was detected on any of the strips. They were detected in pronase-pancreatin digests of the whole iodinated protein but less than 5 % of the radioiodine was present in these forms. In the twelve numbered spots on Fig. 2a with ratios of 1.o7-2.43 (see Table I), all of the radioiodine was present as monoiodotyrosine in the case of eight spots, 95 % as monoiodotyrosine in one spot (No. I5I ), 7 ° % as monoiodotyrosine in another spot (No. 149 ) and 50 % as monoiodotyrosine in two spots (No. 135 and 26). In each spot, any remaining radioactivity was present as diiodotyrosine. Thus, all of these twelve spots were found to contain monoiodotyrosine only or monoiodotyrosine with some diiodotyrosine and none were found which contained only diiodotyrosine. Tile presence of diiodotyrosine in these high-ratio spots apparently represents incomplete separation of monoiodotyrosine and diiodotyrosine peptides. From the thirteen pooled spots with ratios between 1.o5 and 1.24, 78 % of the iodine was present as monoiodotyrosine and 22 % as diiodotyrosine, i.e., the molar ratio of monoiodotyrosine to diiodotyrosine peptides was 7:1. Among the ten individual spots with ratios less than 0.96, three yielded only diiodotyrosine (No. 86, 87 and 89) and the other seven showed both monoiodotyrosine and diiodotyrosine. The presence of both monoiodotyrosine and diiodotyrosine in these spots may be due either to the same behavior in the chromatography and high-voltage paper electrophoresis of both tile monoiodotyrosine peptide and diiodotyrosine peptide derived from a particular tyrosyl residue or to diiodotyrosine and monoiodotyrosine peptides from different sequences migrating together and thus both derivatives being present in the same spot. DISCUSSION
Individual tyrosyl residues in a protein iodinate at different rates. The relative rate of iodination of each tyrosyl residue is dependent on its environment, particularly the exposure of those at the surface of the protein. By using the method described here for measuring the relative rates of iodination of the tyrosyl residues in a protein molecule it is possible to grade these residues in isolated peptides with respect to their relative rates of iodination. These peptides can then be used to identify the tyrosyl residues involved if the amino acid sequence of the protein is known. We have carried out this work with rabbit 7G immunoglobulin primarily because we are interested in the structure of the globulin molecule and of the binding site of the antibody molecule.
The relative rates of iodination of various tyrosines The distribution of the iodine among the various tyrosyl residues of a protein being iodinated depends on the extent of iodination and involves the conversion of Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
7
these residues to monoiodotyrosine or diiodotyrosine residues. This is shown as the successive reactions for any one particular residue. T y r residue + I ~ monoiodotyrosine residue
(i)
Monoiodotyrosine residue + I --~ Diiodotyrosine residue
(2)
where I is the iodinating reagent. The rate equations for the formation of monoiodotyrosine (MIT) and diiodotyrosine (DIT) residues are d(MIT) dt
--
k i ( T ) ( I ) - k~(MIT)(I)
(3)
d(DIT) -- kz(MIT)(I) dt
(4)
where k I and k 2 are specific rate constants for reactions (I) and (2) for the particular residue under consideration. During iodination, the concentration of the iodinating reagent decreases. For each increment of time, dt, each tyrosyl residue is exposed to iodination to the extent of (I)dt where (I) is the instantaneous concentration of the iodinating agent during the interval dr. The total "exposure", z, is then taken as ff(I)dt (see ref. 14). Eqns. (3) and (4) therefore become (5) and (6). d(MIT) dz d(DIT) dr
-
-
-
-
kdZ) -- k2(MIT)
(5)
k2(MIT)
(6)
Integration of (5) and (6) gives Eqns. (7) and (8) (MIT)
kl e-kit k2 - - kl
kl e-k2, ( D I T ) = I -f- - - - - - k2 - - kl
kl e-~.lt k2 - - hi k2 k2
--
(7)
e-kit
(8)
kl
Some tyrosyl residues iodinate faster than others and differences in their remaining concentrations develop during the iodination reaction. In order to illustrate how the relative amounts of monoiodotyrosine and diiodotyrosine derived from various tyrosyl residues of a protein may vary as a function of v, we have chosen the following example of a hypothetical protein for which, (a) the individual molecules of the protein contain 30 iodinatable tyrosyl residues and no other iodinatable group, (b) these residues fall equally into three discrete classes of relative reactivity towards iodination; high, medium and low with relative reactivities 25 : 5 : I and (c) the rate constant for incorporation of a second iodine into an monoiodotyrosine residue is taken to be twice that of the first iodine*. * The considerations which follow hold w h e t h e r k2/k 1 is small or large; only q u a n t i t a t i v e aspects vary. C o n t r a r y to LI's r e p o r t 15 t h a t k 1 is very m u c h larger t h a n k 2, ROCRE et al. TM, MI~NARD a n d SEHON17 and MAYBERRY et al. TM have reported t h a t k~ and k 1 are n o t widely different. Biochim. Biophys. Acta, I47 (1967) 1-14
O. A. ROHOLT, D. PRESSMAN
For the purpose of the calculations, the values of k I for the three classes of reactivity were set at i.o, 0.2 and 0.04 and of k2, at 2.0, 0. 4 and 0.08 with values of T chosen between o and 4o. The relative concentration of monoiodotyrosine and of diiodotyrosine derived from a particular tyrosyl residue as calculated by Eqns. (7) and (8), is shown in Fig. 3. A residue of each class of reactivity (high, medium and low), is represented. I009-
~
_/
~"/-
~
"
TYR
k,
~'MIT
ks
'DIT
08-
al
t-- 0 . 7 -
~'/
~-I
o 067-
j
m
' .I
05<
}
n
P21
........
t.,~'/
,'b ,,,'""
~.~o.?,7,,.'
V , ~
.-,,5.. v ,,'
04-
~_ 0 . 3 -
I--
0.2-
~ , . . : takL..':; 5ff1~ ~ . .IX . . . . . ....... .,, . .3: . . ~" . . . .................... .............. #,
jfll
,.,,,.,.,,,,,
u. 0 1 o
=_.. 0
. ,. . . . .
IT C l a s s 2
I
I
I
I
I
I
5
I0
15
20
25
30
3~
EXPOSURE, I"
Fig. 3. Moles of monoiodotyroslne (MIT) and diiodotyrosine (DIT) from tyrosine residues of three different classes of reactivity plotted against exposure, T, for a model protein with IO tyrosine residues of each class. Their reactivities are in the order 25 : 5 : i w i t h kMIT/kTyr = 2 for each tyrosine.
The number of iodine atoms incorporated into a protein molecule at any value of z is the sum of the iodine incorporated as monoiodotyrosine and as diiodotyrosine at that value of z. Therefore, the number of iodine atoms which would be incorporated per molecule of our hypothetical protein at various values of z have been calculated from the values in Fig. 3 and taking into account that there are ten residues of each class. The relative amount of monoiodotyrosine and diiodotyrosine which would then result from the tyrosyl residues of each reactivity is shown in Fig. 4 as a function of the number of iodine atoms per protein molecule. The amount of monoiodotyrosine (Fig. 4) formed from each tyrosyl residue would pass through a maximum but at a level of iodination dependent on the reactivity of the particular residue. The maximum amount of monoiodotyrosine which could be formed is the same for all the tyrosyl residues in our hypothetical protein because they all have the same ratio of k l / k 2. The amount of diiodotyrosine from each residue would increase and reach saturation at a level of iodination dependent on its reactivity class. The curves in Fig. 5 are derived from Fig. 4 and show the per cent of the total incorporated iodine which would be in the monoiodotyrosine and diiodotyrosine
Biochim. Biophys. Acta,
147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
9
derived from a tyrosyl residue at each class of reactivity as a function of the number of iodine atoms incorporated per protein molecule. At the beginning of the iodination (Fig. 5) all of the iodine would react to give monoiodotyrosine and the per cent of the total iodine present as monoiodotyrosine derived from each individual tyrosyl residue would be 8.1% for one of Class I, 1.6 % for one of Class 2 and 0. 3 % for one of Class 3, proportional to the relative rate constants of the tyrosyl residues concerned, i . e . , 25:5:1. The most reactive tyrosyl residue (Class I) would dominate the picture initially. With increasing levels of iodination, the per cent of iodine as monoiodotyrosine from the most reactive residues would decrease rapidly as those monoiodotyrosine residues are converted to diiodotyrosine residues.
/,,,,,,,,,,,,,,,,,,,,
1.0" M
~'
0.9
~
0.8
~-
0.7'
I1: IAI Z
../
n,.
"
0.6
~.
o.5
,,c
0.4
-'
03
~/
!
o
/
/
..."
2
=E,,,,'.~ ....... ~.....
0.2
Jo
~I
~-" = ~
0 ,, I.-
/
/
-
y ........
0
X
0
IO
20
30
40
50
60
ATOMS r/PROTEIN MOLECULE
Fig. 4- Moles of monoiodotyrosine (MIT) and diiodotyrosine (DIT) trom any particular tyrosine for model protein as in Fig. 3 plotted against atoms of iodine per protein molecule. w
i 7 -[~1 ; 6t\
DIT Class
.. ........
/
~5-~
,:
~ ' o~
~
0
I
.
",.. -.
" I0
~
' 20
~
' 30
°
'
~
40
" 50
60
ATOMS T / PROTEIN MOLECULE
Fig. 5. Per cent of total iodine on each residue for model protein as in Fig. 3 plotted against atoms of iodine per protein molecule. MIT, monoiodotyrosine and DIT, diiodotyrosine.
Biochim. Biophys. Acta, 147 (1967) 1-14
I0
O . A . ROHOLT, D. PRESSMAN
The per cent of iodine incorporated as monoiodotyrosine derived from the tyrosyl residues of medium (Class 2) reactivity would remain low and decrease continuously with increasing iodination, but for the most slowly iodinating tyrosines (Class 3) it would be essentially constant at first and then pass through a maximum. The per cent contribution of the iodine on the diiodotyrosine from each of the most reactive residues to the total incorporated iodine would pass through a maxim u m (7 %) at slightly more than 20 iodine atoms per protein molecule and then decrease (Fig. 5). The per cent of iodine incorporated as diiodotyrosine from each of the residues of intermediate reactivity (Class 2) would increase slowly and pass through a maxim u m of 4 % at about 45 iodine atoms per protein molecule. The per cent of iodine as diiodotyrosine derived from tyrosyl residues of low reactivity (Class 3) would increase rapidly after the iodination level of 4 ° iodine atoms per protein molecule and reach a final m a x i m u m value of 3-3 %, the value at saturation for all the tyrosyl residues.
Peptides containing iodinated tyrosyl residues In the case of the iodination of an actual protein it is probable that each tyrosyl residue will have a different rate of iodination but the curves for the amount of monoiodotyrosine and of diiodotyrosine from a particular tyrosyl residue would still resemble corresponding curves in Figs. 4 and 5. When an iodinated protein is digested by pepsin, various iodinated peptides are obtained. More than one peptide m a y be obtained from a sequence containing a given iodinated tyrosyl residue. The total amount of a peptide obtained containing a particular iodinated tyrosyl residue depends upon the amount of that iodinated residue present in the protein and the mode of splitting by pepsin. The mode of splitting m a y v a r y with the level of iodination and thus influence the amount of some peptides since the pepsin sensitivity of some bonds m a y be altered when the tyrosyl residue has been iodinated 19. In the experimental work presented here, the relative amounts of iodinated peptides formed from two iodinated protein preparations differing in the degree of iodination were precisely compared experimentally by the use of the paired-label technique. One portion of protein was iodinated to a particular level with 131I-labeled iodine, and this preparation was used as a reference. Other portions of the same protein were iodinated to essentially equal or lower levels with 125I-labeled iodine. Individual portions of the reference preparation were then paired (mixed) individually with equal portions of each of the other preparations and the mixture digested by pepsin, thus providing a close control on the digestion and subsequent procedures. In Fig. I a the lalI-labeled reference protein was paired with one iodinated to essentially the same level and the 125I/~S~I ratios for all peptides were the same as that for the unfractionated digest showing that the same amount of any given peptide was derived from each of the two iodinated proteins. This demonstrates the reproducibility of the iodination procedure. In Figs. Ib and ic the l~lI-labeled protein (8.1 iodines per protein molecule) was paired with the proteins iodinated to the levels of 7.4 or 6.4 iodine atoms per molecule. Wide deviations in the ratio of 125I/la~I for m a n y of the peptides were found. These deviations are the basis for determining the relative reactivity of the Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY
OF TYROSYL RESIDUES
TO I O D I N A T I O N
II
various tyrosines in the protein b y this method. Each peptide does not have to be isolated quantitatively to obtain quantitative information about the relative reactivity of the tyrosines. In practice, it is only necessary to isolate the peptide free of other radioiodinated peptides so the ratio can be determined. The relative reactivity of the parent tyrosyl residue determines whether the ratio for a peptide is greater or less than the ratio for the original mixture, and whether the peptide is a monoiodotyrosine or diiodotyrosine peptide. These values tell whether the parent residue is just beginning to be iodinated at the level of iodination used, or is being actively iodinated or has been already saturated with iodine. The theoretical basis for deducing the extent of iodination of a tyrosyl residue follows. The illustrative curves shown in Fig. 6 were chosen to show how the relative amounts of monoiodotyrosine formed at different levels of iodination depends on the reactivity of the parent tyrosyl residue. These curves are of the form derived in Fig. 4 and represent the relative amount of monoiodotyrosine formed as a function of the level of iodination of the protein for each of five tyrosyl residues of different reactivity (Fig. 6). The most reactive tyrosyl residue is represented in Fig. 6a and the least reactive in Fig. 6e with those of three intermediate activities in Fig. 6b, c and d. The two vertical lines labeled "low" and "high" indicate the two levels of iodination used. The points of interception of these lines and each monoiodotyrosine curve represent the relative amounts of monoiodotyrosine formed in each case. In Fig. 6a the tyrosine is so reactive that the maximal amount of monoiodotyrosine is formed at a level of iodination which is lower than either level used. In Fig. 6b, for a slightly less reactive tyrosine, the high level still exceeds the level of maximal monoiodotyrosine formation but the low level corresponds to it. In Fig. 6c, for a residue of intermediate reactivity, the two levels straddle the level of maximal monoiodotyrosine formation so that nearly equal amounts of monoiodotyrosine are formed in each case. In Fig. 6d the high level is at the level of maximal monoiodotyrosine formation and in Fig. 6e the tyrosyl residue is of such low reactivity relative to others that the curve is concave upward prior to reaching its maximum. On the right side of Fig. 6, there is the ratio of the amounts of monoiodotyrosine formed from these tyrosyl residues at the two indicated levels of iodination of the protein. This ratio is greater than unity for the most reactive residue and less than unity for the least reactive with the others falling in between these values. The ratio drops to less than the quotient of the number of iodine atoms per protein molecule at the low level of iodination divided by the number at the high level only for the least reactive residues (Fig. 6e) where the forepart of the curve is concave upward. The diiodotyrosine peptides are dealt with a parallel manner in Fig. 7 using curves of the form derived in Fig. 4. The ratio of the amount of a given diiodotyrosine peptide from the protein iodinated at the low level to that at the high level will be equal to unity for only the most reactive residues since they are saturated (Fig. 7a). I t will be less than unity for all others. The value of this ratio m a y be greater than, equal to, or less than the value of the quotient of the number of iodine atoms per protein molecule at the low level and the number at the high level. If the diiodotyrosine curve at the low level is above the dotted line, the ratio for the peptide is greater than the quotient of the two levels of iodination of the protein and if below the dotted line, it is less. Biochim. Biophys. Acta, 147 (1967) i - I 4
12
O . A . ROHOLT, D. PRESSMAN
From these considerations, information about a number of relationships for peptides containing only one iodinated tyrosyl residue can be derived from a diagram such as Fig. I on which the ratio of the two levels of iodination of the protein is shown as a solid horizontal line. They hold even if several peptides are derived from the same sequence. In analyzing data as in Fig. I the following conclusions are useful. LEVEL OF IODINATION LEVEL OF IODINATION LOW
HIGH
LOW
RATIO PEPTIDE FROM LOW LEVEL HIGH •
HIGH
RATIO PEPTIDE FROM LOW LEVEL " " HIGH "
7
>>t, >>R
~I,
>R
Y (c)
<1 ~:
(c) .
(d)
ATOMS OF IODINE PER PROTEIN MOLECULE
(e)
I
od,
/ < I
, <:R
R .ol..N MOLECO. E
Fig. 6. Curves illustrating the theoretical relationship between the n u m b e r of a t o m s of iodine incorporated per protein molecule and the relative a m o u n t s of monoiodotyrosine (MIT) formed from tyrosine residues of decreasing reactivities t o w a r d iodination. R is the q u o t i e n t of the n u m b e r of a t o m s of iodine per protein molecule at the low level divided by the n u m b e r at the high level of iodination. The dotted line is the locus of the values of this q u o t i e n t for a n y low level of iodination. See t e x t for discussion. Fig. 7- Same as Fig. 6 b u t for diiodotyrosine (DIT).
a. All peptides with a ratio greater than one must represent monoiodotyrosine peptides (see Fig. 6) and the ratio for a diiodotyrosine peptide always has a value of one or less (see Fig. 7). This is verified b y the data of the experiment shown in Fig. 2. In accord with this conclusion, 12 spots with a ratio greater than one were analyzed and found to contain monoiodotyrosine. Nearly all contained only monoiodotyrosine. Where diiodotyrosine was found, there is reason to believe that this was due to incomplete resolution of peptides. Furthermore, all of the spots which were found to contain only diiodotyrosine had a ratio of less than one. b. Information concerning the reactivity of different tyrosyl residues toward iodination can be obtained as follows: (i) A monoiodotyrosine peptide with a ratio greater than one must come from a more rapidly iodinating tyrosine ; the level of iodination of the more highly iodinated protein is already greater than that at which the maximal amount of this monoiodotyrosine peptide is formed (see Fig. 6a, b). Therefore, in the experiment represented in Fig. 2, the peptides in the 9 spots with ratios greater than 1.25 (Table I) were from Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
13
more rapidly iodinating tyrosines. They were shown to be monoiodotyrosine peptides. (ii) A monoiodotyrosine peptide with a ratio of one means that the parent tyrosine is one which is in the process of undergoing active iodination at the levels of iodination being used (see Fig. 6c). Spots No. 15o, 151 and 152 from the experiment in Fig. 2 had ratios of 1.o7-1.16 and were essentially all monoiodotyrosine peptides and were thus derived from tyrosines undergoing active iodination. (iii) A monoiodotyrosine peptide with a ratio less than tile ratio of the iodination levels of the proteins, i.e., a ratio falling below the solid line, must come from a slowly reacting tyrosine (see Fig. 6e). (iv) A diiodotyrosine peptide with a ratio of one and which is present in a large amount means that the tyrosine residue is a more rapidly iodinating tyrosine and has been iodinated to saturation even at the lower level of iodination used (Fig. 7a). (v) All diiodotyrosine peptides falling above the solid line come from tyrosyl residues which are almost saturated in the more highly iodinated protein and which are therefore iodinated more rapidly than the tyrosines giving rise to diiodotyrosine peptides falling below the solid line (Fig. 7b). Thus, for the experiment in Fig. 2, peptides No. 86, 87 and 89 are from tyrosines almost saturated with iodine at the higher level. (vi) Diiodotyrosine peptides in high proportion and below the solid line represent tyrosines of intermediate reactivity in the process of undergoing active iodination to diiodotyrosine (see Fig. 7c). (vii) Diiodotyrosine peptides showing the lowest values for the ratio are from the less reactive tyrosine residues which are just entering the active phase of iodination from monoiodotyrosine to diiodotyrosine (see Fig. 7 d, e). c. The extent to which an amino acid sequence containing a particular tyrosyl residue (iodinated) may yield several different peptides in the enzymatic hydrolysis is shown on Fig. 2 as follows: (i) Peptides with the same ratio, but appreciably different from tile average, probably come from tile same sequence although not necessarily so. Thus peptide 26 and peptides 28 and 29 may arise from the same sequence. (ii) When monoiodotyrosine peptides having ratios of approximately one are present in relatively small amounts they must be derived from one or more tyrosinecontaining sequences which have been split into several peptides each containing the tyrosine. Otherwise, they would be present in larger amounts (Fig. 6c). (iii) When diiodotyrosine peptides fall above the solid line and are in relatively small amounts, they must be derived from a tyrosine-containing sequence which has been split into several peptides. Otherwise they would have to be present in larger amounts (Fig. 7 a, b). d. Principles for the design of additional experiments to obtain data about particular tyrosine residues follow directly from exploratory experiments. The yield of particular peptides may be increased by the following: (i) A diiodotyrosine peptide with a ratio less than one will increase in amount upon further iodination of the low level protein (Fig. 7b-e). (ii) A monoiodotyrosine peptide with a ratio of one or less will initially increase in amount on further iodination of the less iodinated protein (Fig. 6d, e). Experimentally this method for determining the relative rates of iodination of Biochim. Biophys. Acta, 147 (1967) 1-14
14
O . A . ROHOLT, D. PRESSMAN
the various tyrosines has the advantage over the iodination with a single iodine isotope in that quantitative recovery of each peptide is not required as when the single isotope is used. Relative values are obtained directly from each spot. The analysis does, however, depend on the isolation of the peptide free from other iodinated peptides; losses are not important, and the ratio is of utmost significance. Assigning relative rates of iodination to specific tyrosyl residues of a protein requires that each tyrosyl residue can be identified from the amino acid sequence of the peptide in which it is present. Then the relative reactivity of a residue, as determined by this method of paired iodination using the above conclusions, can be associated with a particular residue identified by sequence analysis of the peptide and knowledge of the amino sequence of the protein. In the case of those proteins for which the crystal structure and sequence has been determined, examination of the structure along with application of our procedure might show some correlation between the two with regard to the apparent exposure of the tyrosines and their relative reactivity toward iodination as determined by this procedure. ACKNOWLEDGEMENTS
We wish to thank Mr. GERALD RADZIMSKI,Mr. DAVID PARK and Mr. JOSEPH GURRERI, Jr., for technical assistance. This research was supported in part by Grant AIo3962 from the National Institute of Allergy and Infectious Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19
D. PRESSMAN AND L. A. STERNBERGER, J. Immunol., 66 (1951) 6o 9. A. JOHNSON, E. D. DAY AND D. PRESSMAN, J. Immunol., 84 (196o) 213. A. L. GROSSBERG, G. RADZlMSKI AND D. PRESSMAN, Biochemistry, I (1962) 391. R. W. WOODY, M. E. FRIEDMAN AND H. A. SCHERAGA, Biochemistry, 5 (1966) 2034. P. H. SPRINGELL, Nature, 191 (1961) 1372. L. W. DE ZOETEN AND O. A. DE BRUIN, Rec. Tray. Chim. Pays-Has, 80 (1961) 9o 7. T. T. HERSKOVlTS AND M. LASKOWSKI, Jr., J . Biol. Chem., 237 (1962) 2481. S. I{. DUBE, O. A. ROHOLT AND D. PRESSMAN, J . Biol. Chem., 241 (1966) 4665. D. PRESSMAN AND O. A. ROHOLT, Proc. Natl. Acad. Sci., 47 (1961) 16o6. O. A. ~R.OHOLT, A. SHAW AND D. PRESSMAN, Nature, 196 (1962) 773. R. R. PORTER, Biochem. J., 73 (1959) 119. P. STELOS, O. A. ROHOLT AND D. PRESSMAN, J. Immunol., 89 (1962) 113. R. BIRD AND H. E. A. FARRAN, J. Clin. Endoerinol. Metab., 20 (196o) 81. S. WIDEQVlST, Arkiv Kemi, 8 (1956) 545. C. H. LI, J. Am. Chem. Soc., 64 (1942 ) 1147. J. ROCHE, S. LlSSlTZKY, O. MICHEL AND R. MICHEL, Biochim. Biophys. Acta, 7 (1951) 439. C. MENARD AND A. H. SEHON, Abstr, Sept. MeetingAm. Chem. Soc., 5957, 24S. W. E. MAYBERRY, J. E. RALL, M. BERMAN AND D. BERTOLI, Biochemistry, 4 (1965) 1965L. E. BAKER, J. Biol. Chem., 193 (1951) 809.
Biochim. Biophys. Acta, 147 (1967) 1-14
I
BBA 3 5 1 1 2
A DIFFERENTIAL METHOD FOR DETERMINING THE RELATIVE R E A C T I V I T Y TO I O D I N A T I O N OF D I F F E R E N T T Y R O S Y L R E S I D U E S IN A P R O T E I N MOLECULE O. A. R O H O L T AND D. P R E S S M A N
Department of Biochemistry Research, Roswell Park Memorial Institute*, Buffalo, N.Y. (U.S.A.) (Received May 22nd, 1967)
SUMMARY
(I) A method has been developed for determining the relative rates of iodination of various tyrosyl residues in a protein. This is done b y the use of a paired-label technique in which one portion of the protein is iodinated with iodine labeled with 125I and another portion is iodinated to incorporate a different number of iodine atoms labeled with 131I. The proteins are mixed and enzymatically digested. The resulting iodinated peptides are separated. Each iodinated peptide separated is a mixture of the 131I-labeled peptide from the first iodination and the 125I-labeled peptide from the second iodination. (2) The relative reactivities of particular tyrosyl residues (identified by surrounding sequences) toward iodination can be determined from an analysis of the ratios of the amounts of iodine incorporated in the corresponding tyrosyl peptides derived from each of the two iodinated preparations. (3) There is no need to recover quantitatively all the monoiodotyrosine and diiodotyrosine peptides derived from a particular tyrosine, a recovery which is difficult since for a single tyrosyl residue both of these forms can be distributed among several peptides.
INTRODUCTION
All of the various tyrosyl residues in a protein molecule do not seem to be exposed to the same degree. Experiments involving the iodination of several antibodies have shown that the iodination of tyrosyl residues is largely responsible for a loss of antibody activity and that the tyrosyl in the combining site seems to be iodinated at a rate more rapid than the average rate for all tyrosyl residues l-a. In the case of ribonuclease, it is relatively easy to iodinate some of the tyrosyl residues while others are iodinated very slowly under the same conditions 4. Of the tyrosyl residues in insulin, those of the A chain are iodinated in the intact molecule more readily than those of the B chain according to SPRINGELL~ and DE ZOETEN AND DE BRUIN6. They have taken this to mean that different tyrosyl residues are exposed to different extents. In the case of ribonuclease and bovine serum albumin, the ab* A u n i t of the New Y o r k State D e p a r t m e n t of Health.
Biochim. Biophys. Acta, 147 (1967) 1-14
2
o . A . ROHOLT, D. PRESSMAN
sorption spectrum varies for the different tyrosyl residues and this also indicates different exposure 7. We have found that the reactivities of the four tyrosyl residues of native ~-chymotrypsin towards iodination are in the order 146 > 94 > 171 > 229 with relative reactivities of I : 0.4: 0.2: 0.02, respectivelys. We have studied the relative rates of iodination of different residues in a protein and have been able to isolate peptides containing tyrosyl residues of differing reactivities toward iodination. This was done by a paired-label technique9,1°, where one portion of the protein was iodinated to a certain level with iodine labeled with 125I and another portion was iodinated to a somewhat higher level of iodination with iodine containing la1I. These two iodinated preparations were then mixed and digested with pepsin and the resulting peptides were separated by high-voltage paper electrophoresis and chromatography. Differences in the amounts of each iodinated peptide as determined from the two iodine isotopes indicated the relative degrees of reactivity towards iodination of various tyrosyl residues in the protein. The protein studied was a portion of the antibody molecule which had been isolated following papain digestion of the antibody molecule and which was of particular interest since it contains the combining site".
MATERIALS AND METHODS
Antibody fraction A papain digest 11 of specifically purified rabbit anti-/5-azobenzoate antibody" was chromatographed on CM-cellulose and the fraction designated as I I c in our previous reporO 2 was used.
Enzymes Pronase (enzyme P from Streptomyces griseus) was obtained from Biddle Sawyer Company, New York, N.Y. and pancreatin from Merck and Company, Rahway, N.J.
Iodination of protein Iodinations were carried out as described previously using hypoiodite labeled with either 1251 or 1311 (see ref. 9)-
Isolation of iodinated ])eptides Mixtures of 1251- and 131I-labeled protein in 0.5 M HCOOH were digested with pepsin (protein-pepsin, 30: I, by weight) at 37 ° for 15-2o h. The digests were subjected to chromatography (n-butanol-acetic acid-water, 4:1:5, by vol.,) on W h a t m a n 3 MM paper followed by high-voltage paper electrophoresis (High Voltage Electrophoretor, Model D, Gilson Medical Electronics). The separations were carried out at 40 V per cm using 4 % HCOOH as the background electrolyte. Radioautographs of the dried paper were prepared b y exposing it to Kodak Industrial X - R a y Film Type KK1 for suitable periods. Spots on the radioautograph were sequentially numbered and the area on the paper corresponding to each spot was cut out and the 1251/131I ratio for each spot was calculated after counting in an automatic recording, dual-channel g a m m a - r a y spectrometer. Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
3
RESULTS
Variations in the amount of iodinated peptide obtained following peptic digestion of a protein iodinated to different levels Three 2.5-ml portions of a solution containing 4.1 mg of the protein were each exposed to a different concentration of hypoiodite labeled with 125I. The level of iodine incorporation was 6. 4 , 7.4 and 8. 4 iodine atoms per antibody fragment (50000, tool. wt.). A fourth portion was iodinated to the level of 8.1 iodine atoms per fragment with lslI-labeled iodine, and served as the reference protein against which the others were compared. A portion of each of the three l~SI-labeled proteins was individually mixed with an equal weight of the lS~I-labeled protein to give three paired mixtures. The paired mixtures were digested with pepsin. Portions of the digest were subjected to paper chromatography and to high-voltage paper electrophoresis and radioautographs were prepared. The patterns of spots on the radioautographs were similar since they were all due primarily to the ~31I label of the single preparation of reference protein above. Corresponding spots on each radioautograph were readily recognized*. All the spots ATOMS I
PER MOLECULE
8.4 (125T), 8.[ (131I) (0)
,:oE 1.5[
z
Z4 (125I), 8.t (131I)
H .J
1'5 f 1.0 0.8 0.6
I5
34 13
:r?o
'
L,
25
l''"
,b,
30
mH 2.0
14
r
I
IO
t
34
8 TTT
T .
64 (t25 I) I~
8"I (t311)
(c)
19
,T T,
l,
T
50
I 000
t
I
I0 000 13[I (COUNTS PER MINUTE PER SPOT )
I00 000
Fig, I. Relative a m o u n t s of iodinated peptides obtained from F r a c t i o n I I c of an anti-X~ a n t i b o d y following iodination to different levels. E a c h p o i n t represents the results for an individual spot cut from the electrophoretogram. The o r d i n a t e shows the ratio of the a m o u n t of 12SI-labeled iodine to the a m o u n t of 131I-labeled iodine, i.e., the ratio of the a m o u n t s of a given iodinated peptide derived from each of the t w o iodinated protein p r e p a r a t i o n s t h a t were mixed and digested. On the abscissa, the 1311 c o u n t per s p o t is shown. * E v e n t h o u g h the same a m o u n t of the same lSlI-labeled protein was t a k e n for (a), (b) or (c) of Fig. i, the c o u n t rate of 1811 observed for a given spot m a y n o t be the same due to variations in the area of the s p o t cut from the p a p e r ; this does n o t affect the value of the ratio for the spot, however, unless the s p o t is a poorly resolved mixture.
Biochim. Biophys. Acta, 147 (1967) 1-14
4
O . A . R O H O L T , D. PRESSMAN
were numbered for identification and comparison. Some of the nmnbers are given on Fig. I. The ratio of the amount of ~25I-labeled iodine to the amount of 131I-labeled iodine was then determined for each spot. This ratio is equivalent to the ratio of the amount of the iodinated peptide obtained from each of the paired proteins. Ratios for the three paired proteins are shown in Fig. I. The solid line represents the quotient of the number of iodine atoms per protein molecule at the low level of iodination and the number at the high level of iodination. In the case of the pair for which the level of iodination was nearly the same, 8.4 and 8.1 iodine atoms per molecule, the amount of each of the radioactive peptides obtained from each of the proteins was the same (Fig. Ia). In the other mixtures, where the differences in the levels of iodination were greater (Fig. ib, c), the ratios for m a n y of the iodinated peptides deviated rather widely from one another.
Determination of the presence of monoiodotyrosine and of diiodotyrosine in the isolated, iodinated peptides In another experiment, 5 mg of the antibody protein was iodinated with i25Ilabeled iodine and a second portion was iodinated with l~lI-labeled iodine but to a higher level. The level of iodine incorporation was found to be 6.6 4 iodine atoms per protein molecule for the 125I-labeled material and 7.63 for the ~3~I-labeled material. Equal weights of the two were mixed and digested with pepsin. I mg of the digested protein was subjected to chromatography followed by high-voltage paper electrophoresis and a radioautograph was prepared. The ratio of 125I-labeled iodine to ~31I-1abeled iodine was determined for each spot as shown in Figs. 2a, b. The ratios for selected spots are shown in Table I. The presence of monoiodotyrosine or of diiodotyrosine in the peptide associated with these selected spots was then determined. Twelve spots with ratios greater than 1.o5, including nine spots with the highest ratios (1.25-2.43) and three of the most TABLE
I
SPOTS SELECTED
FROM PATTERN
S H O W N O N P I G . 23, F O R A N A L Y S I S O F M O N O - A N D D I I O D O T Y R O S I N E
Analyzed individually Ratio ~ z.o5 Spot No.
Ratio
26 28 29 51 53 66 74 135 149 15o 151 152
1.97 2.13 1.87 1-47 2.43 1.66 1.5° 1.52 1.25 i.io 1.o 7 1.16
Pooled for analysis Ratio ~ 0.96
125I
(counts~rain) I 060 2 600 9 lo 5 2oo 13 3 ° 0 3 600 2 920 2 760 6 760 18 800 22 800 22 200
Spot No.
Ratio
32 54 55 56 57 69 83 86 87 89
0.64 0.82 o.81 0.82 0.86 0.76 0.72 0.92 0.96 0.96
Ratio I.o5-I.24 12~I
(counts~rain)
Biochim. Biophys. ,4cta, 147 (1967) I 14
i 24 19 io 9 8 2 17 19 19
250 400 ooo 300 020 090 OlO 300 700 200
Spot No.
Ratio
43 52 73 118 132 146 147 148 158 159 165 166 167
I.O9 1.24 1.o 9 1.17 1.09 1.16 I.IO 1.o 9 1.13 1.22 1.31
125I (counts/rain) 7 67o 45 ° I 12o 47 ° 4 800 2 960 6 ooo 12 600 3 34 ° 860 I 320
1.21
I 600
1.2o
I 480
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
5
radioactive spots (ratios 1.o7-1.16 ) were individually analyzed for monoiodotyrosine and diiodotyrosine. Additionally, ten spots with ratios less than o.96 were individually analyzed for monoiodotyrosine and diiodotyrosine. Thirteen other spots with ratios between 1.o5 and 1.24 were treated as a pool (see Table I).
ATOMS "r PER MOLECULE 6.6 ( } z s I ) , 7.6( 131"r ) H
3I
2O .2 H
10 08
,,~?,
,'
r!
~ -,r,.
'X~
0 o
101000
100 131I (COUNTS PER MINUTE PER SPOT)
Fig. 2a. R a d i o a u t o g r a p h of iodinated peptides separated from a peptic digest of iodinated antib o d y f r a g m e n t b y c h r o m a t o g r a p h y a n d high-voltage paper electrophoresis. Peptides eluted from the spots indicated b y arrows were analyzed for t h e presence of mono- or diiodotyrosine. The q u a n t i t a t i v e d a t a concerning each of t h e spots are in Table I. Fig. 2b. Same as Fig. I b u t for a n o t h e r preparation of antibody.
The analyses were carried out by pulping the paper in minimal volumes of i M NH4OH and the pulp filtered on a flitted funnel and then similarly extracted two more times. Over 95 % of the radioactivity was recovered in a volume of less than 2 ml. The eluates were lyophilized in Io-ml erlenmeyer flasks. Hydrolysis of the peptides was done b y adding 0. 4 ml of 0.05 M ammonium bicarbonate containing o.I mg of pronase to each flask and incubating at 37 ° for 4 h. The contents were lyophilized and a further digestion using pancreatin was then carried out in the same manner. The digests were again lyophilized and the residues taken up in minimal volumes of I M formic acid containing carrier monoiodotyrosine, diiodotyrosine, Biochirn. Biophys. Acta, 147 (I967) 1-14
6
O. A. ROHOLT, D. PRESSMAN
monoiodohistidine and diiodohistidine. This was applied to 1.25 inch Whatman 3 MM strips and subjected to high-voltage paper electrophoresis in I M formic acid in order to separate the iodinated compound. Radioautographs were then prepared. Areas on the paper which corresponded to the radioactivity were cut out and counted. The paper strips were reconstituted and the positions of all of the iodinated compounds located by means of a ceric sulfate-arsenious acid spray 13. Comparison of the sprayed strip and the radioautograph permitted an unambiguous identification of the radioactive material as monoiodotyrosine or as diiodotyrosine. Neither radioiodinated monoiodohistidine nor diiodohistidine was detected on any of the strips. They were detected in pronase-pancreatin digests of the whole iodinated protein but less than 5 % of the radioiodine was present in these forms. In the twelve numbered spots on Fig. 2a with ratios of 1.o7-2.43 (see Table I), all of the radioiodine was present as monoiodotyrosine in the case of eight spots, 95 % as monoiodotyrosine in one spot (No. I5I ), 7 ° % as monoiodotyrosine in another spot (No. 149 ) and 50 % as monoiodotyrosine in two spots (No. 135 and 26). In each spot, any remaining radioactivity was present as diiodotyrosine. Thus, all of these twelve spots were found to contain monoiodotyrosine only or monoiodotyrosine with some diiodotyrosine and none were found which contained only diiodotyrosine. Tile presence of diiodotyrosine in these high-ratio spots apparently represents incomplete separation of monoiodotyrosine and diiodotyrosine peptides. From the thirteen pooled spots with ratios between 1.o5 and 1.24, 78 % of the iodine was present as monoiodotyrosine and 22 % as diiodotyrosine, i.e., the molar ratio of monoiodotyrosine to diiodotyrosine peptides was 7:1. Among the ten individual spots with ratios less than 0.96, three yielded only diiodotyrosine (No. 86, 87 and 89) and the other seven showed both monoiodotyrosine and diiodotyrosine. The presence of both monoiodotyrosine and diiodotyrosine in these spots may be due either to the same behavior in the chromatography and high-voltage paper electrophoresis of both tile monoiodotyrosine peptide and diiodotyrosine peptide derived from a particular tyrosyl residue or to diiodotyrosine and monoiodotyrosine peptides from different sequences migrating together and thus both derivatives being present in the same spot. DISCUSSION
Individual tyrosyl residues in a protein iodinate at different rates. The relative rate of iodination of each tyrosyl residue is dependent on its environment, particularly the exposure of those at the surface of the protein. By using the method described here for measuring the relative rates of iodination of the tyrosyl residues in a protein molecule it is possible to grade these residues in isolated peptides with respect to their relative rates of iodination. These peptides can then be used to identify the tyrosyl residues involved if the amino acid sequence of the protein is known. We have carried out this work with rabbit 7G immunoglobulin primarily because we are interested in the structure of the globulin molecule and of the binding site of the antibody molecule.
The relative rates of iodination of various tyrosines The distribution of the iodine among the various tyrosyl residues of a protein being iodinated depends on the extent of iodination and involves the conversion of Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
7
these residues to monoiodotyrosine or diiodotyrosine residues. This is shown as the successive reactions for any one particular residue. T y r residue + I ~ monoiodotyrosine residue
(i)
Monoiodotyrosine residue + I --~ Diiodotyrosine residue
(2)
where I is the iodinating reagent. The rate equations for the formation of monoiodotyrosine (MIT) and diiodotyrosine (DIT) residues are d(MIT) dt
--
k i ( T ) ( I ) - k~(MIT)(I)
(3)
d(DIT) -- kz(MIT)(I) dt
(4)
where k I and k 2 are specific rate constants for reactions (I) and (2) for the particular residue under consideration. During iodination, the concentration of the iodinating reagent decreases. For each increment of time, dt, each tyrosyl residue is exposed to iodination to the extent of (I)dt where (I) is the instantaneous concentration of the iodinating agent during the interval dr. The total "exposure", z, is then taken as ff(I)dt (see ref. 14). Eqns. (3) and (4) therefore become (5) and (6). d(MIT) dz d(DIT) dr
-
-
-
-
kdZ) -- k2(MIT)
(5)
k2(MIT)
(6)
Integration of (5) and (6) gives Eqns. (7) and (8) (MIT)
kl e-kit k2 - - kl
kl e-k2, ( D I T ) = I -f- - - - - - k2 - - kl
kl e-~.lt k2 - - hi k2 k2
--
(7)
e-kit
(8)
kl
Some tyrosyl residues iodinate faster than others and differences in their remaining concentrations develop during the iodination reaction. In order to illustrate how the relative amounts of monoiodotyrosine and diiodotyrosine derived from various tyrosyl residues of a protein may vary as a function of v, we have chosen the following example of a hypothetical protein for which, (a) the individual molecules of the protein contain 30 iodinatable tyrosyl residues and no other iodinatable group, (b) these residues fall equally into three discrete classes of relative reactivity towards iodination; high, medium and low with relative reactivities 25 : 5 : I and (c) the rate constant for incorporation of a second iodine into an monoiodotyrosine residue is taken to be twice that of the first iodine*. * The considerations which follow hold w h e t h e r k2/k 1 is small or large; only q u a n t i t a t i v e aspects vary. C o n t r a r y to LI's r e p o r t 15 t h a t k 1 is very m u c h larger t h a n k 2, ROCRE et al. TM, MI~NARD a n d SEHON17 and MAYBERRY et al. TM have reported t h a t k~ and k 1 are n o t widely different. Biochim. Biophys. Acta, I47 (1967) 1-14
O. A. ROHOLT, D. PRESSMAN
For the purpose of the calculations, the values of k I for the three classes of reactivity were set at i.o, 0.2 and 0.04 and of k2, at 2.0, 0. 4 and 0.08 with values of T chosen between o and 4o. The relative concentration of monoiodotyrosine and of diiodotyrosine derived from a particular tyrosyl residue as calculated by Eqns. (7) and (8), is shown in Fig. 3. A residue of each class of reactivity (high, medium and low), is represented. I009-
~
_/
~"/-
~
"
TYR
k,
~'MIT
ks
'DIT
08-
al
t-- 0 . 7 -
~'/
~-I
o 067-
j
m
' .I
05<
}
n
P21
........
t.,~'/
,'b ,,,'""
~.~o.?,7,,.'
V , ~
.-,,5.. v ,,'
04-
~_ 0 . 3 -
I--
0.2-
~ , . . : takL..':; 5ff1~ ~ . .IX . . . . . ....... .,, . .3: . . ~" . . . .................... .............. #,
jfll
,.,,,.,.,,,,,
u. 0 1 o
=_.. 0
. ,. . . . .
IT C l a s s 2
I
I
I
I
I
I
5
I0
15
20
25
30
3~
EXPOSURE, I"
Fig. 3. Moles of monoiodotyroslne (MIT) and diiodotyrosine (DIT) from tyrosine residues of three different classes of reactivity plotted against exposure, T, for a model protein with IO tyrosine residues of each class. Their reactivities are in the order 25 : 5 : i w i t h kMIT/kTyr = 2 for each tyrosine.
The number of iodine atoms incorporated into a protein molecule at any value of z is the sum of the iodine incorporated as monoiodotyrosine and as diiodotyrosine at that value of z. Therefore, the number of iodine atoms which would be incorporated per molecule of our hypothetical protein at various values of z have been calculated from the values in Fig. 3 and taking into account that there are ten residues of each class. The relative amount of monoiodotyrosine and diiodotyrosine which would then result from the tyrosyl residues of each reactivity is shown in Fig. 4 as a function of the number of iodine atoms per protein molecule. The amount of monoiodotyrosine (Fig. 4) formed from each tyrosyl residue would pass through a maximum but at a level of iodination dependent on the reactivity of the particular residue. The maximum amount of monoiodotyrosine which could be formed is the same for all the tyrosyl residues in our hypothetical protein because they all have the same ratio of k l / k 2. The amount of diiodotyrosine from each residue would increase and reach saturation at a level of iodination dependent on its reactivity class. The curves in Fig. 5 are derived from Fig. 4 and show the per cent of the total incorporated iodine which would be in the monoiodotyrosine and diiodotyrosine
Biochim. Biophys. Acta,
147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
9
derived from a tyrosyl residue at each class of reactivity as a function of the number of iodine atoms incorporated per protein molecule. At the beginning of the iodination (Fig. 5) all of the iodine would react to give monoiodotyrosine and the per cent of the total iodine present as monoiodotyrosine derived from each individual tyrosyl residue would be 8.1% for one of Class I, 1.6 % for one of Class 2 and 0. 3 % for one of Class 3, proportional to the relative rate constants of the tyrosyl residues concerned, i . e . , 25:5:1. The most reactive tyrosyl residue (Class I) would dominate the picture initially. With increasing levels of iodination, the per cent of iodine as monoiodotyrosine from the most reactive residues would decrease rapidly as those monoiodotyrosine residues are converted to diiodotyrosine residues.
/,,,,,,,,,,,,,,,,,,,,
1.0" M
~'
0.9
~
0.8
~-
0.7'
I1: IAI Z
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"
0.6
~.
o.5
,,c
0.4
-'
03
~/
!
o
/
/
..."
2
=E,,,,'.~ ....... ~.....
0.2
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~I
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0 ,, I.-
/
/
-
y ........
0
X
0
IO
20
30
40
50
60
ATOMS r/PROTEIN MOLECULE
Fig. 4- Moles of monoiodotyrosine (MIT) and diiodotyrosine (DIT) trom any particular tyrosine for model protein as in Fig. 3 plotted against atoms of iodine per protein molecule. w
i 7 -[~1 ; 6t\
DIT Class
.. ........
/
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,:
~ ' o~
~
0
I
.
",.. -.
" I0
~
' 20
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' 30
°
'
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40
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60
ATOMS T / PROTEIN MOLECULE
Fig. 5. Per cent of total iodine on each residue for model protein as in Fig. 3 plotted against atoms of iodine per protein molecule. MIT, monoiodotyrosine and DIT, diiodotyrosine.
Biochim. Biophys. Acta, 147 (1967) 1-14
I0
O . A . ROHOLT, D. PRESSMAN
The per cent of iodine incorporated as monoiodotyrosine derived from the tyrosyl residues of medium (Class 2) reactivity would remain low and decrease continuously with increasing iodination, but for the most slowly iodinating tyrosines (Class 3) it would be essentially constant at first and then pass through a maximum. The per cent contribution of the iodine on the diiodotyrosine from each of the most reactive residues to the total incorporated iodine would pass through a maxim u m (7 %) at slightly more than 20 iodine atoms per protein molecule and then decrease (Fig. 5). The per cent of iodine incorporated as diiodotyrosine from each of the residues of intermediate reactivity (Class 2) would increase slowly and pass through a maxim u m of 4 % at about 45 iodine atoms per protein molecule. The per cent of iodine as diiodotyrosine derived from tyrosyl residues of low reactivity (Class 3) would increase rapidly after the iodination level of 4 ° iodine atoms per protein molecule and reach a final m a x i m u m value of 3-3 %, the value at saturation for all the tyrosyl residues.
Peptides containing iodinated tyrosyl residues In the case of the iodination of an actual protein it is probable that each tyrosyl residue will have a different rate of iodination but the curves for the amount of monoiodotyrosine and of diiodotyrosine from a particular tyrosyl residue would still resemble corresponding curves in Figs. 4 and 5. When an iodinated protein is digested by pepsin, various iodinated peptides are obtained. More than one peptide m a y be obtained from a sequence containing a given iodinated tyrosyl residue. The total amount of a peptide obtained containing a particular iodinated tyrosyl residue depends upon the amount of that iodinated residue present in the protein and the mode of splitting by pepsin. The mode of splitting m a y v a r y with the level of iodination and thus influence the amount of some peptides since the pepsin sensitivity of some bonds m a y be altered when the tyrosyl residue has been iodinated 19. In the experimental work presented here, the relative amounts of iodinated peptides formed from two iodinated protein preparations differing in the degree of iodination were precisely compared experimentally by the use of the paired-label technique. One portion of protein was iodinated to a particular level with 131I-labeled iodine, and this preparation was used as a reference. Other portions of the same protein were iodinated to essentially equal or lower levels with 125I-labeled iodine. Individual portions of the reference preparation were then paired (mixed) individually with equal portions of each of the other preparations and the mixture digested by pepsin, thus providing a close control on the digestion and subsequent procedures. In Fig. I a the lalI-labeled reference protein was paired with one iodinated to essentially the same level and the 125I/~S~I ratios for all peptides were the same as that for the unfractionated digest showing that the same amount of any given peptide was derived from each of the two iodinated proteins. This demonstrates the reproducibility of the iodination procedure. In Figs. Ib and ic the l~lI-labeled protein (8.1 iodines per protein molecule) was paired with the proteins iodinated to the levels of 7.4 or 6.4 iodine atoms per molecule. Wide deviations in the ratio of 125I/la~I for m a n y of the peptides were found. These deviations are the basis for determining the relative reactivity of the Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY
OF TYROSYL RESIDUES
TO I O D I N A T I O N
II
various tyrosines in the protein b y this method. Each peptide does not have to be isolated quantitatively to obtain quantitative information about the relative reactivity of the tyrosines. In practice, it is only necessary to isolate the peptide free of other radioiodinated peptides so the ratio can be determined. The relative reactivity of the parent tyrosyl residue determines whether the ratio for a peptide is greater or less than the ratio for the original mixture, and whether the peptide is a monoiodotyrosine or diiodotyrosine peptide. These values tell whether the parent residue is just beginning to be iodinated at the level of iodination used, or is being actively iodinated or has been already saturated with iodine. The theoretical basis for deducing the extent of iodination of a tyrosyl residue follows. The illustrative curves shown in Fig. 6 were chosen to show how the relative amounts of monoiodotyrosine formed at different levels of iodination depends on the reactivity of the parent tyrosyl residue. These curves are of the form derived in Fig. 4 and represent the relative amount of monoiodotyrosine formed as a function of the level of iodination of the protein for each of five tyrosyl residues of different reactivity (Fig. 6). The most reactive tyrosyl residue is represented in Fig. 6a and the least reactive in Fig. 6e with those of three intermediate activities in Fig. 6b, c and d. The two vertical lines labeled "low" and "high" indicate the two levels of iodination used. The points of interception of these lines and each monoiodotyrosine curve represent the relative amounts of monoiodotyrosine formed in each case. In Fig. 6a the tyrosine is so reactive that the maximal amount of monoiodotyrosine is formed at a level of iodination which is lower than either level used. In Fig. 6b, for a slightly less reactive tyrosine, the high level still exceeds the level of maximal monoiodotyrosine formation but the low level corresponds to it. In Fig. 6c, for a residue of intermediate reactivity, the two levels straddle the level of maximal monoiodotyrosine formation so that nearly equal amounts of monoiodotyrosine are formed in each case. In Fig. 6d the high level is at the level of maximal monoiodotyrosine formation and in Fig. 6e the tyrosyl residue is of such low reactivity relative to others that the curve is concave upward prior to reaching its maximum. On the right side of Fig. 6, there is the ratio of the amounts of monoiodotyrosine formed from these tyrosyl residues at the two indicated levels of iodination of the protein. This ratio is greater than unity for the most reactive residue and less than unity for the least reactive with the others falling in between these values. The ratio drops to less than the quotient of the number of iodine atoms per protein molecule at the low level of iodination divided by the number at the high level only for the least reactive residues (Fig. 6e) where the forepart of the curve is concave upward. The diiodotyrosine peptides are dealt with a parallel manner in Fig. 7 using curves of the form derived in Fig. 4. The ratio of the amount of a given diiodotyrosine peptide from the protein iodinated at the low level to that at the high level will be equal to unity for only the most reactive residues since they are saturated (Fig. 7a). I t will be less than unity for all others. The value of this ratio m a y be greater than, equal to, or less than the value of the quotient of the number of iodine atoms per protein molecule at the low level and the number at the high level. If the diiodotyrosine curve at the low level is above the dotted line, the ratio for the peptide is greater than the quotient of the two levels of iodination of the protein and if below the dotted line, it is less. Biochim. Biophys. Acta, 147 (1967) i - I 4
12
O . A . ROHOLT, D. PRESSMAN
From these considerations, information about a number of relationships for peptides containing only one iodinated tyrosyl residue can be derived from a diagram such as Fig. I on which the ratio of the two levels of iodination of the protein is shown as a solid horizontal line. They hold even if several peptides are derived from the same sequence. In analyzing data as in Fig. I the following conclusions are useful. LEVEL OF IODINATION LEVEL OF IODINATION LOW
HIGH
LOW
RATIO PEPTIDE FROM LOW LEVEL HIGH •
HIGH
RATIO PEPTIDE FROM LOW LEVEL " " HIGH "
7
>>t, >>R
~I,
>R
Y (c)
<1 ~:
(c) .
(d)
ATOMS OF IODINE PER PROTEIN MOLECULE
(e)
I
od,
/ < I
, <:R
R .ol..N MOLECO. E
Fig. 6. Curves illustrating the theoretical relationship between the n u m b e r of a t o m s of iodine incorporated per protein molecule and the relative a m o u n t s of monoiodotyrosine (MIT) formed from tyrosine residues of decreasing reactivities t o w a r d iodination. R is the q u o t i e n t of the n u m b e r of a t o m s of iodine per protein molecule at the low level divided by the n u m b e r at the high level of iodination. The dotted line is the locus of the values of this q u o t i e n t for a n y low level of iodination. See t e x t for discussion. Fig. 7- Same as Fig. 6 b u t for diiodotyrosine (DIT).
a. All peptides with a ratio greater than one must represent monoiodotyrosine peptides (see Fig. 6) and the ratio for a diiodotyrosine peptide always has a value of one or less (see Fig. 7). This is verified b y the data of the experiment shown in Fig. 2. In accord with this conclusion, 12 spots with a ratio greater than one were analyzed and found to contain monoiodotyrosine. Nearly all contained only monoiodotyrosine. Where diiodotyrosine was found, there is reason to believe that this was due to incomplete resolution of peptides. Furthermore, all of the spots which were found to contain only diiodotyrosine had a ratio of less than one. b. Information concerning the reactivity of different tyrosyl residues toward iodination can be obtained as follows: (i) A monoiodotyrosine peptide with a ratio greater than one must come from a more rapidly iodinating tyrosine ; the level of iodination of the more highly iodinated protein is already greater than that at which the maximal amount of this monoiodotyrosine peptide is formed (see Fig. 6a, b). Therefore, in the experiment represented in Fig. 2, the peptides in the 9 spots with ratios greater than 1.25 (Table I) were from Biochim. Biophys. Acta, 147 (1967) 1-14
REACTIVITY OF TYROSYL RESIDUES TO IODINATION
13
more rapidly iodinating tyrosines. They were shown to be monoiodotyrosine peptides. (ii) A monoiodotyrosine peptide with a ratio of one means that the parent tyrosine is one which is in the process of undergoing active iodination at the levels of iodination being used (see Fig. 6c). Spots No. 15o, 151 and 152 from the experiment in Fig. 2 had ratios of 1.o7-1.16 and were essentially all monoiodotyrosine peptides and were thus derived from tyrosines undergoing active iodination. (iii) A monoiodotyrosine peptide with a ratio less than tile ratio of the iodination levels of the proteins, i.e., a ratio falling below the solid line, must come from a slowly reacting tyrosine (see Fig. 6e). (iv) A diiodotyrosine peptide with a ratio of one and which is present in a large amount means that the tyrosine residue is a more rapidly iodinating tyrosine and has been iodinated to saturation even at the lower level of iodination used (Fig. 7a). (v) All diiodotyrosine peptides falling above the solid line come from tyrosyl residues which are almost saturated in the more highly iodinated protein and which are therefore iodinated more rapidly than the tyrosines giving rise to diiodotyrosine peptides falling below the solid line (Fig. 7b). Thus, for the experiment in Fig. 2, peptides No. 86, 87 and 89 are from tyrosines almost saturated with iodine at the higher level. (vi) Diiodotyrosine peptides in high proportion and below the solid line represent tyrosines of intermediate reactivity in the process of undergoing active iodination to diiodotyrosine (see Fig. 7c). (vii) Diiodotyrosine peptides showing the lowest values for the ratio are from the less reactive tyrosine residues which are just entering the active phase of iodination from monoiodotyrosine to diiodotyrosine (see Fig. 7 d, e). c. The extent to which an amino acid sequence containing a particular tyrosyl residue (iodinated) may yield several different peptides in the enzymatic hydrolysis is shown on Fig. 2 as follows: (i) Peptides with the same ratio, but appreciably different from tile average, probably come from tile same sequence although not necessarily so. Thus peptide 26 and peptides 28 and 29 may arise from the same sequence. (ii) When monoiodotyrosine peptides having ratios of approximately one are present in relatively small amounts they must be derived from one or more tyrosinecontaining sequences which have been split into several peptides each containing the tyrosine. Otherwise, they would be present in larger amounts (Fig. 6c). (iii) When diiodotyrosine peptides fall above the solid line and are in relatively small amounts, they must be derived from a tyrosine-containing sequence which has been split into several peptides. Otherwise they would have to be present in larger amounts (Fig. 7 a, b). d. Principles for the design of additional experiments to obtain data about particular tyrosine residues follow directly from exploratory experiments. The yield of particular peptides may be increased by the following: (i) A diiodotyrosine peptide with a ratio less than one will increase in amount upon further iodination of the low level protein (Fig. 7b-e). (ii) A monoiodotyrosine peptide with a ratio of one or less will initially increase in amount on further iodination of the less iodinated protein (Fig. 6d, e). Experimentally this method for determining the relative rates of iodination of Biochim. Biophys. Acta, 147 (1967) 1-14
14
O . A . ROHOLT, D. PRESSMAN
the various tyrosines has the advantage over the iodination with a single iodine isotope in that quantitative recovery of each peptide is not required as when the single isotope is used. Relative values are obtained directly from each spot. The analysis does, however, depend on the isolation of the peptide free from other iodinated peptides; losses are not important, and the ratio is of utmost significance. Assigning relative rates of iodination to specific tyrosyl residues of a protein requires that each tyrosyl residue can be identified from the amino acid sequence of the peptide in which it is present. Then the relative reactivity of a residue, as determined by this method of paired iodination using the above conclusions, can be associated with a particular residue identified by sequence analysis of the peptide and knowledge of the amino sequence of the protein. In the case of those proteins for which the crystal structure and sequence has been determined, examination of the structure along with application of our procedure might show some correlation between the two with regard to the apparent exposure of the tyrosines and their relative reactivity toward iodination as determined by this procedure. ACKNOWLEDGEMENTS
We wish to thank Mr. GERALD RADZIMSKI,Mr. DAVID PARK and Mr. JOSEPH GURRERI, Jr., for technical assistance. This research was supported in part by Grant AIo3962 from the National Institute of Allergy and Infectious Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19
D. PRESSMAN AND L. A. STERNBERGER, J. Immunol., 66 (1951) 6o 9. A. JOHNSON, E. D. DAY AND D. PRESSMAN, J. Immunol., 84 (196o) 213. A. L. GROSSBERG, G. RADZlMSKI AND D. PRESSMAN, Biochemistry, I (1962) 391. R. W. WOODY, M. E. FRIEDMAN AND H. A. SCHERAGA, Biochemistry, 5 (1966) 2034. P. H. SPRINGELL, Nature, 191 (1961) 1372. L. W. DE ZOETEN AND O. A. DE BRUIN, Rec. Tray. Chim. Pays-Has, 80 (1961) 9o 7. T. T. HERSKOVlTS AND M. LASKOWSKI, Jr., J . Biol. Chem., 237 (1962) 2481. S. I{. DUBE, O. A. ROHOLT AND D. PRESSMAN, J . Biol. Chem., 241 (1966) 4665. D. PRESSMAN AND O. A. ROHOLT, Proc. Natl. Acad. Sci., 47 (1961) 16o6. O. A. ~R.OHOLT, A. SHAW AND D. PRESSMAN, Nature, 196 (1962) 773. R. R. PORTER, Biochem. J., 73 (1959) 119. P. STELOS, O. A. ROHOLT AND D. PRESSMAN, J. Immunol., 89 (1962) 113. R. BIRD AND H. E. A. FARRAN, J. Clin. Endoerinol. Metab., 20 (196o) 81. S. WIDEQVlST, Arkiv Kemi, 8 (1956) 545. C. H. LI, J. Am. Chem. Soc., 64 (1942 ) 1147. J. ROCHE, S. LlSSlTZKY, O. MICHEL AND R. MICHEL, Biochim. Biophys. Acta, 7 (1951) 439. C. MENARD AND A. H. SEHON, Abstr, Sept. MeetingAm. Chem. Soc., 5957, 24S. W. E. MAYBERRY, J. E. RALL, M. BERMAN AND D. BERTOLI, Biochemistry, 4 (1965) 1965L. E. BAKER, J. Biol. Chem., 193 (1951) 809.
Biochim. Biophys. Acta, 147 (1967) 1-14