Kinetic and thermodynamic studies of antigen-antibody interactions in heterogeneous phase reaction systems—II.

016l-5890/79/0601-0379

SO2.@3/0

KINETIC AND THERMODYNAMIC STUDIES OF ANTIGENANTIBODY INTERACTIONS IN HETEROGENEOUS PHASE REACTION SYSTEMS-II. INTERACTION

ANTIBODY

(L-T,)WITH SPECIFIC ON CONTROLLED-PORE GLASS

OF L-TRIIODOTHYRONINE

1MMOBILIZED W. HERTL

and G. ODSTRCHEL

Corning Glass Works, Sullivan Science Park. Research and Development Labs, Corning, NY 14830, U.S.A.

Abstract-The binding of triiodothyronine (T,) to its specific antibody immobilized on a porous inorganic support (eontrolled-pore glass) was treated ktnetically as a general heterogeneous reaction. The reactions follow apparent second order kinetics, where the rate is proportional to the amount of antibody and to the instantaneous concentration of available unbound Tj: this unbound T; is inversely related to the concentrations of those serum proteins which normally bind T,. In the presence of these proteins T, diffusion is not the rate determining step of the reaction. For reaction times in hr the extent of the T, binding is inversely related to the protein concentration and the reaction rate does not change greatly with varying protein concentration. For reaction times of weeks the presence or absence of protein or deblocking agent has no effect on the amount of T. bound. but the reaction rates are inversely related to the protein concentration. The function of %a&inonaphthaiene suifonic acid (ANS) debiocking agent is to effectively displace the T, from the TBG fraction of the proteins. which has no effect on the equilibrium point but greatly accelerates the reaction rate. The rate determining step is believed to be the rate of desotption of T; from the protein. since the T, binding rate to antibody takes place rapidly. The measured enthaipy of the reaction (O-1.3 kcal/moi) indicates that the reaction is principally entropy driven.

INTRODUCTION

was carried out to identify the rate dctermini~g step of the reaction sequence, and then this step was studied more closely.

A previous report (Hertl & Odstrchel, 1978) described the kinetic and thermodynamic properties of the interaction of L-thyroxine (L-T,) with its specific antibody immobiIi~~ on controlled-pore glass particles (CPG). L-T~, although principally made at

MATERIALSANDMETHODS Materiai.s

the target tissue rather than being secreted by the thyroid directly, coexists in serum with L-T, at a concentration that is lo2 times lower than L-T+. Like r4 it is distributed between albumin, prealbumln and thyroxine binding globulin (TBG), but with L-T; the association constants with these three proteins are significantly lower than T,. Nevertheless, the measurement of L-T~ in human serum samples by radioimmunoassay (RIA) requires the presence of a chemical deblocking agent [e.g. thimerosal. Sanilinonaphthalene sulfonic acid (ANS)] to free it from the proteins (Chopra, 19721, thus ensuring accurate evaluation of t-T, concentrat~ons.Whether the effect of these chemical agents is due solely to a shift in the equilibrium distribution of the Tj between the proteins and the antibody, or also affects the rate of binding to the antibody has not been experimentally verified. This report deals with a study of the fundamental kinetic and thermodynamic factors which control the binding rates of L-T; to its specific immobilized antibody. The binding of T, to its antibody under various conditons was treated in terms of a general

heterogeneous reaction. A kinetic analysis of the data

L-T; was obtained from Sigma Chemical Company. Radiolabeled L-T; was obtained from Corning Medicai Products. Medfield, Massachusetts. The specific activity was 500-I 200 j.K;#lg. Preparation

oj L-7_ immunogen and anti,sera

L-T~ was coupled to BSA by use of CPI water-soluble carbodiimide (I-cyclohexyi-3-(2-morph~linoethyl~carbodiimide metho-n-sulfate, Aldrich Chemical Co.) and antisera raised by the method of Gharib PI al. (1971). Analysis for iodine yielded a molar ratio of 4-10 moles of LT, to 1.Omole of BSA. Bleedings were taken from the central artery of the ear and the titer evaluated in a wet titer system using 50?$ saturated ammonium sulfate to separate bound and free labeled antigen (Goodfriend & Odya, 1974). Antisera specific for L-T~ was immobilized covalently on controlled pore glass (CPG) and titered as described by Herti and Odstrchel (1978). These porous glass particles have a mean particle diameter of 1.7 {trn and a surface area of 80 mZ/g. Cross

reacting substances

Thecrossreactivity of the antibody for a substance may be measured as the ratio of the amount of T, required to 379

380

W. HERTL Table

I. Cross reactivity

of immobilized

and G. ODSTRCHEI

antibody

Ratlo of Cone required CTOSSfor 50”,, displacement (ngiml) reactivity

Substance

tixed solution bolurnc m an Ostnald densities. necessary for calculating measured in a pycnometer bottle.

RESULTS

I-triiodothyrosme d-triiodothyronine

(T,)

l-thyroxine (T,) d-thyroxine tetraiodothyroacetic triiodothyroacetic monoiodotyrosme diiodotyrosine

acid

acid

2.0 42 I I50 700 I x0

1.00 0.048 0.0017 0.0029 0.01 I

41

0.049 0

> IO” > IO”

asprin

> IO”

5,5_diphenylhydantoin phenylbutazone methimazole 6-N-propyl-2-thiouracll

> IO” > IO” > IO” > IO”

0 0 0 0 0 0

displace 50”) of the T, label from antl-T,; to the amount ot cross-reacting substance to give hke displacement. The measured cross-reactivities are given in Table I. Preparation

of serum protein

Pooled human serum wascharcoal extracted to remove T; and T, (Hollander & Shenkmen. 1974). Residual TZ was below the detection limit as determlned by Corning IMMO PHASETU T; assay kit. Theextracted serum was lyophilized. This lyophilized serum was reconstituted in distilled water to the desired protem concentration. The protein concentration was checked spectrophotometrically after carrying out the Biuret reactjon (Fahey & Terry. 1967).

.ACD DISCUSSION

These reactions will bc treated in terms of a general heterogeneous reaction. in which the soluble antigen, T3. binds to its specific antibody that is immobilized on porous glass particles. Thus, the minimum

sequence

of steps which must take place is:

(1) desorption ofT; from any proteins to which it is bound; (2) diffusion of T, through the liquid phase; and (3) adsorption/desorption of T; with respect to its

antibody.

A large number ofreaction curves were obtained for the binding of T; in buffer solution. buffer-ANS, and with various levels of serum proteins both with and without added ANS. To quantify the observed reaction rates for comparison. the following treatment was used on some of the data. Various simplified rate equations were used to test the data. until reasonably good linear plots were obtained. An apparent second order intergrated rate equation of the following form gave the best tit:

kt =

.Y

11

TBG was extracted from pooled serum and purified by the method described by Marshall. Pensky & Williams (1973). The TBG free serum was also retalncd.

where:

(u

.Y )

(I = the ‘endpoint’ or extent of the reaction: .Y = the fraction of T3 bound, at any time =r; h- = the apparent

To perform a solid-phase T,; radiolmmunoassay the various constituents (IMA. serum. ANS. labeled T,; and PBS) are added to a tube and agitated. After a given time (typical.ly I hr) the tube i, centrifuged causing the immoblhzed antibody to pellet in the bottom ofthe tube. The number of radioactive counts from the pellet are measured. The per cent of Tj* bound to the IMA with respect to the total amount ofT,* is then calculated. A constant correction due to the small amount of T,; adsorbed on the wall of the tube is also made.

For kinetic experiments it is necessary to accurately control the reaction. or incubation time. The details for the procedure of obtaining reaction curves have been previously given by Hertl and Odstrchel(l978). Briefly. the constituents are added to the tube. allowed to react for the desired time and then centrifuged to stop the reaction. The number of counts are measured in the pelleted IMA and in the supernatant liquid. from which the fraction of radioactive Tj* bound to the antibody is calculated. This procedure is carried out with a series of tuber which are allowed to react for different periods of time. from which one obtains the fraction T;* bound as a function of time. In any concurrent series of experiments only one parameter was varied at a time. such as protein concentration or viscosity All mcasurments were obtained in duplicate

For studying diffusion effects. solutions of differmg viscosities were made by adding sucrose to PBS buffer. The viscosities weredetermined by measuring thedescent time ofa

L’l\cometcr. Solurlon the vl\cosil!. were

rate constant.

An example of some reaction curves and the kinetic plots obtained is shown below in Figs. 4 and 5. A nicely linear plot results. The slope of this plot gives the value of the apparent rate constant which is a quantitative measure of the binding reaction rate under the conditions of the particular experiment. Due to variations in different lots of immobilized antibody. the exact values of the reaction rates should only be compared within a given series of concurrent experiments. D~fjusion

in the liquid phusc~

If diffusion through the liquid phase is the ratedetermining step of the overall reaction sequence, then the reaction rate will be proportional to the diffusion coefficient of the diffusing species. T;. The diffusion coefficient can be easily varied by changing the viscosity (PJ)of the liquid medium, since the diffusion coefficient is inversely proportional to the viscosity. The fraction of T3 bound in a fixed period of time with respect to the total that binds is a measure of the reaction velocity. so that if the reaction is diffusion controlled. these reaction velocities will vary as l/q. This was tested experimentally by making up sucrose solutions in buffer having different viscosities. The results are given in Fig. I and are plotted as the fraction of- the reaction with respect to the amount of

T, Binding IO

381

Kinetics

I

I% SERUM/BUFFER ,T, 8 3% SERUM/BUFFER

,( 02 02

06

04

08

IO

15

30

“7

Fig. I. Effect of viscosity on binding rates. The ratio of the amount of binding at the given viscosity to the amount of binding measured at I .Ocs is plotted against the reciprocal of the viscosity. All tubes contained 0.050 ml 0.25 ng/ml standard T,. The measured viscosities of the solutions containing the 0.050 ml of I and 8”< serum protein are indicated. in buffer solution (defined as 1.00) against l/V Over the range of viscosities used in this model system, the reaction is clearly diffusion controlled. Also on the plot are simultaneously measured T; binding rates measured in the presence of 1 and 8 per cent added serum protein. The rates are inhibited to a much greater extent than can be accounted for by the viscosity of the liquid medium. Thus, in the presence of serum proteins the reaction rate is not controlled by diffusion through the liquid phase. reaction

Efjtict of’ serum proteins on the reaction

It has been observed that serum proteins inhibit the apparent extent of the T; binding reaction. In the T; assay ANS is used to ‘deblock’ the T, from the binding protein(s), and, therefore, the extent of reaction both with and without added ANS were measured. In Fig.2 the data obtained for the per cent T; bound in the presence of various concentrations of serum proteins are plotted. The points were measured after 96-120 hr to ensure that the reaction had reached a pseudo-equilibrium point. It is seen that the extent of

0 SERUM IANS 0 ” ” OSERUM

I

0

I1 2

96 HRS 120 HRS

ONLY (NO ANSI

11

4 % PROTEIN

45

60

1 (MINUTES1

I 6 IN ADDED

I1

1

8

I IO

Fig. 3. Kinetic plots in system containing various concentrations of 0.050 ml added serum proteins. End points measured at 5 hr.

reaction decreases linearly with increasing protein concentration. Comparison of the closed points (added ANS) with the open points (no added ANS) shows that after relatively long reaction times (in this case 96-120 hr) the per cent T; bound reaches approximately the same level. Figure 3 gives some typical kinetic plots taken from a series of reaction curves using 0, 1.05and 8.4 per cent serum protein, all with ANS. The measured values of the apparent rate constants are given in Table 2 along with rate data from other experiments. The values of ‘a’, the ‘end point,’ were typically measured after 5 hr reaction, a point at which the reaction curve is very flat. Comparison of the data given in Table 2 shows that the highest rate constants are observed in buffer only solutions; as soon as any ANS or serum is present in the system the rate constants drop by about one half. There is no significant difference in rate constants with differing levels of serum with ANS. The kinetic Table 2. Protein effects: measured rates System

k(min

Buffer-ANS I”:, serum-ANS 8% serum-ANS

0.2 I 0.13 0.14

Buffer” Buffer-ANY 1.05% serum-AN?? 8.4$ Serum/ANY

0.27 0.15 0.13 0.12

Buffer Buffer-ANS

0.39 0.20

Buffer 8.4’: 0 serum-ANS

0.15 0.07

Buffer Buffer-ANS

0.27 0.12

I)

SERUM

Fig. 2. Extents of binding in solutions containing various concentrations of 0.050 ml added serum protein, with and without 5 x 10m4 M ANS. Extents of binding measured at 96120 hr.

“cf. Fig. 3 for kinetic plots. End points measured at 5 hr; all with 0.5 ng/ml standard T, added. ANS. when used is 5 x 10m4 M.

3x2

W. HERTL

and G. ODSTRCHEL

iI:

I% SERUM

f Fig. 4. Reaction

(min)

’

RI,,,> serum ’

The kinetic data indicated

I%

SERUM

PROTEIN/ANS

8%

SERUM

PROTEIN/BUFFER

(hrs)

&,,

,erum-ANS

> Rw,> xru,,,.

Identification qf’ interfering proteins

14

8%

curves in system containing various concentrations ofO.050 ml added berum proteins and without 5 x IO 1 M ANS.

significance of obtaining the same rates with various serum protein concentrations is that the ANS displaces the T; from the proteins at about the same rate. If one now allows the reaction to take place for extremely long times (600 hr) the effect of the protein on the extent of reaction is drastically minimized and approaches the values obtained in the absence of protein. In Fig. 4 are given some reaction curves using I and 8 per cent serum both with and without added ANS. The data from Fig.4 are given as kinetic plots in Fig. 5. The data in Figs. 2 and 3 (Table 2) show that for reaction times up to about 100 hr the extent of reaction is depressed with increasing protein concentration and the reaction rate to this meta stable equilibrium point is little affected by the presence of serum protein with ANS. The rates, however, are slower than in the complete absence of protein and ANS. After extended reaction times (600 hr, Figs. 4 and 5) the extent of reaction is independent of the serum protein and ANS concentrations, but the rate of approach to this true equilibrium point is inversely related to the protein concentration and proceeds faster in the presence of ANS. The data in Fig. 8 shows that the relative rates of reaction are: R I”,,wlumANS

PROTEIN/ANS

that there are two classes

with

of interfering proteins. i.e. one class in which ANS deblocks the T? and another class not affected by ANS. To posltlvely identify these proteins, known quantities of thyroxine binding gloublin (TBG) were added to TBG-free serum. A set of reaction curves is shown in Fig. 6. It is clear that the presence of TBG inhibits the reaction rate; addition of ANS gives a rate similar to that observed in the TBG free serum. Clearly, the protein affected by the ANS is the TBG. Prealbumin and albumin form the other class of proteins little affected by ANS. and requiring hundreds of hr for all the T; to desorb. The binding rates in the absence of protein are sufficiently fast for the binding of T_: to its antibody not to be the rate determining step of the reaction sequence in the presence of protein. If the desorption rate of T, from the binding proteins is the rate determining step rather than the adsorption rate on the IMA then one should be able to minimize the protein interference by performing a ‘biased’ experiment. To this end, the constituents were added to the tubes in different orders and the binding measured after 15min. The results are given in Table 3. In the buffer system, the T; is immediately available for binding to the IMA since no proteins are present. When the Tj is added it is all available for binding to the IMA until the protein is present. When the serum is added first, the Tj adsorbs on both the protein and IMA, but then has to desorb from the protein to adsorb on the IMA, and this is a slow process. These data, then. are consistent with a model in

PROTEIN/AN

12

0

IO

20

30

40

50 60 + (MINUTES)

Fig. 5. Kinetic plots of data in Fig. 4. End points measured 652 hr. ANS. when used. is 5 x IOmJ M.

120

at

Fig. 6. Reaction curves obtained In protcm free system. In system with added TBG and 5 x IO ~4 A4 ANS. and in system with added TBG only.

383

T; Binding Kinetics 60

Table 3. Effect of order of addition on reaction rate Order

BUFFER

10 6ml

IMA

2 ml IMA

/ANS/O

a

46.8 32.6 6.1

O,,T; bound min.

measured

the rate determining

after

IS

‘I

‘I

step is the desorption

of the

Comparison of deblocking agents

It is of interest to compare the effect of the deblocking agent ANS with that of merthiolate used in a previous study (Hertl & Odstrchel, 1978). The binding reactions of both T; and T, with their specific antibodies were measured. Since these are different systems the values of the binding rates cannot be meaningfully compared other than to note that in both systems the absence of a deblocking agent gives low binding rates and with either ANS or merthiolate present the binding rate is much faster. The extents of binding within each system, however, can be compared and the data are given in Table 4. As noted above, in the T, system the presence or absence of ANS has little effect on the equilibrium extent of binding. With merthiolate a small additional quantity of T; is displaced from one of the proteins. With T,, the previously observed effect of shifting the equilibrium somewhat is again observed when merthiolate is used; with ANS the shift in equilibrium is less. Since the deblocking effect of ANS was shown above to be principally on the TBG, these data suggest that merthiolate also displaces T, and T, from the albumin and/or TBPA. Effect of antibody concentration Although it is reasonable to assume that one of the concentration dependent terms is the available concentration of antibody (IMA), experiments were carried out to confirm this. Two concurrent series were run, one of which contained three times the normal concentration of IMA. The protein and ANS concentrations were adjusted so that the same extent of binding was obtained after 5 hr. The reaction curves obtained are given in Fig. 7 where one sees that with the higher IMA concentration one obtains a substantially faster reaction rate. This confirms that of ANS and merthiolate agents

J 300

h

u

Fig. 7. Reaction curves obtained with two different antibody concentrations. Serum concentrations were chosen to obtain same extent of binding at 5 hr.

the antibody concentration is one of the concentration dependent terms in the rate equation. Temperature dependence Reaction tubes were made up with buffer only, with serum protein, and with serum-ANS. The tubes with serum also contained 0.050 ml 0.5 ng/ml standard T;. Since preliminary data showed that the reactions are very slow at low termperatures, the tubes were all incubated at 23°C for five days and then samples were stored at SC, 23°C and 38°C. The per cent Tj bound was measured at weekly intervals. After 400 hr, constant binding values were attained. As previously observed, the presence or absence of ANS had little effect on the final equilibrium point. The data obtained at 480 hr are given on the van’tHoff plot in Fig. 8. A very slight effect due to the proteins is observable on the plot; in buffer only the enthalpy of adsorption is 1.3 Kcal/mole, with serum present the measured enthalpy is O-O.7 Kcal/mole. These very small values for enthalpies of adsorption show that the major contribution to the free energy of binding (Table 5) is due to the entropy change. This is in accord with literature data for haptene-antibody reactions (Keane et al., 1976). CONCLUSION

These kinetic data show that diffusion is not important in determining the rates of the T,JIMA reactions. The T; adsorption rate on IMA is quite fast, but the desorption rate from the two types of proteins is slow. With proteins of the first type (TBG) the rate

nH

deblocking I

i

, 3 Kca~

BUFFER ONLY

SERUM/AN

a-

al 9 D

Deblocking agent

System

I I IO 15 t (MINUTES1

I 5

0

T; from the proteins.

Table 4. Comparison

c

PROTEIN/ PROTEIN

‘9, Tj bound

of addition

Buffer-T, T,-serum Serum-T,

which

50

0 5% 0 I%

0

Yn

a”=O-07Kcal tl

SERUM

_B

P

I6T, (0.25 ngjml std T,, 4”. serum protem. 5 x IO-” M ANS or merthmlate)

NOW ANS Merthmlale

OC 23

38

I 33

T, (40 ng!ml std T,. 4% serum protem, 5 * IO-’ MANS or merthmlate)

NolIe ANS Merthmlate

Extents

of reaction

measured

after two weeks.

720”, 79.5”, 92 79,

I 34 ICOO/T

8

I 35

Fig. 8. Extents of binding measured at three temperatures in systems without serum, with serum, and with serum-5 x 10m4 MANS. The slope of this van tHoff plot is a measure of the enthalpy of reaction.

3x4

W. HERTL

and G. ODSTRC‘HEI

Table 5. Binding constants for T.; system K

Binding component

A G,,

Thyroxine binding globulin”

6.5 x IO’ 12.0 Kcal;mole

Pre-albuminh Albumin’ TBG-ANSd IMA

1.2 3.X 4.2 I.3

x IO* IO’ x IO” x IO”’

9.7 Kcal/molc 7.6 Kcal;mole 9.0 Kcal:molc 13.7 Kcal’mole

“Kureck c’t ~1.. 1976 ‘Pages el (11.. 1973. ’ Steiner (‘t al., 1966. ClGreen et al.. 1972.

of desorption can be effectively accelerated by adding ANS. Although ANS has a somewhat lower free energy of binding to the protein than T3 (Table 5), it is present in very large molar excess and thus effectively displaces the T,. With proteins of the second type (albumin, prealbumin) the rate of desorption is extremely slow and little affected by ANS. The reactions follow second order kinetics in which the rate of reaction is determined by the concentrations of IMA sites and of instantaneously available free T;, this latter quantity being determined by the rate of desorption from the protein during the course of the reaction. Calculation shows that in serum the greater part (>99”{,) of the T3 exists as a complex with the proteins. The kinetic significance of this is that in the T3-IMA system as each Tj binds to an IMA site, (the T; being that small fraction of truly ‘free T,‘) it is necessary for T3 to desorb from the proteins to which they are bound to try to restore the equilibrtum relationships. Thus, the free T, is being replenished to the solution phase from the adsorbed phase. Although T, and T; have similar ampholytic properties, the three dimensional structure of these molecules differs significantly. T, is more planar in nature than T;, which appears crystallographically to have a greater dihedral angle between the two benzene like rings. One would not be surprised then if significant differences were observed in the interaction with their respective antibodies upon release from binder proteins. In fact, this is not the case in these systems. The kinetic as well as the thermodynamic data do not greatly differ. Both of these reactions are

entropy driven and the observed differences can be ascribed to differences in the binding energies of T, and T, to serum proteins. .~~,Xllolr/c,~/~c,n~c~,,r.\The Lluthors gratefully acknowledge the as\istancc of E. R. Herritt 111carrying OUI the cxperlmental work

REFERE\CES

Chopra I. J.. Ho R. S. & Ldm R. (1971) An improved radioimmunoassay of trllodothyronme, Its application to chnical and physiologvzal utudw ./. f.crh dirr. ,~cd. 80. 729.

Gharlb H.. Ryan R. H. & Maqbcrry W. E (1971) Radioimmunoassay for trllodothyroninc (T;): Affinity and specificity of antihodq for T;. .I. ~/in. O&C r. :Mctcrh 33, 509. Goodfriend T. & Odya C‘. E. (I 974) ,M~rllr~/.\ CJ/ /~or~~rorw Rudi~~r,tln~u,lot.s,\~~l,(Edited by Jaffee B. M. Nr Bchrman H. R.) p. 439. Academic Pres. Nc\r York. Green A. M.. Marshall J. S.. Pensky J. & Stanbury J B ( 1972) Thyroxine bindmg globulm. characterization of the blnding \Itc uith lluorcvzent dye a\ ;I probe. .Sc~icwc~~ 175. 137x Hcrtl W. & Odstrchel G. (I 97X) Kmcrlc and thermodynamtc studies ofantigen-antibody interactIon\ in hetcrogeneou\ reaction phases 1. Mulct ~/trr /nrwww/o~,. 16, 173-l 7X. Hollander C. S. & Shcnkmen L. ( 1974) Mctko& of Hownonc~ Ruc/~r,ir,lrnrmou.~.c~~~.(Edltcd by Jaffc B. I. & Behrman H. R. p.)715. Academic Press. Nem York. Kureck L. & Tabachnick V (1976) Thyrovnc protein interactionsofthyrowineand triiodothqroninc with human thyroxine binding globuhn. ./. hr/~/ C$I~I 251. 3.55X Kcane P. M.. Walker W H.. Gauldie J. & Abraham G. b. (1976) Thermodynamic aspects of some radioasa!,. C‘/III. (‘lr1w1. 22, 70. Marshall J. S.. Pcnsky J. & Wllllam~ S. (1973) Studw in human thyroxine hindlng globulin VIII Iwelectrlc focusing nnmunoheterogenicity of thyroxine binding globulin. Arc/~ /~ioc~/wn Biopl~>,.. 156, 456. Pages R. A.. Robbins J. & Edelhoch H. (1973) Binding of thyroxine and thyroxine analogs to human wrum prealbumln. Biw/wnr.~r,-v 12, 2773. Steiner R. I-‘.. Roth J. & Robhint J. (1966) The blnding 01 thyroxine by serum albumin as mcasurcd bj tluoresccncc quenching. J. hioc /GUI. C‘lww. 241. 560.