Immune precipitation of purified chicken antibody at low pH

Immunochemistry. PergamonPress. 1970. Vol.7, pp. 771-785. Printed in Great Britain

I M M U N E P R E C I P I T A T I O N OF PURIFIED CHICKEN

ANTIBODY AT LOW pH* JOAN S. GALLAGHER and EDWARD W. VOSS, Jr. Department of Microbiology, University of Illinois, Urbana, Ill. 61803, U.S.A. (First received 2 March 1970; in revisedform 14 Apr/11970) A b s t r a c t - I n c r e a s e d immune precipitation of purified chicken anti-DNP antibody with homologous antigen at physiological salt concentrations was measured by lowering the pH to 5.0. Ultracentrifuge studies indicated that at pH 5-0 the antibody molecule was not aggregated and existed as a monomer with a $20,,oof 6.75 and a molecular weight o f 172,600. Electrofocusing profiles of chicken anti-DNP antibody gave a pI value of

6"6 for the monomeric form. Binding studies of chicken anti-DNP by fluorescence quenching indicated that pH 5.0 had little or no effect on the direct interaction between antigen and antibody. Equilibrium dialysis studies with 3H-~-DNP-L-lysine at pH 5.0 gave an average intrinsic association constant of 1.7 × 106M -1 identical to that at pH 8"0. The number of ligand binding sites per antibody molecule, based on a molecular weight of 172,600 was 2"0. INTRODUCTION Proposed explanations for the inability of the salt requiring chicken IgG antibody to precipitate with homologous antigen in low salt (NaCI) have been univalency[1, 2] and a low average intrinsic association constant (K0)[2]. However, recent results showed that purified chicken anti-2,4-DNP antibody was bivalent and had a K0 of 1"7 × 106M-113] in both low and high concentrations of NaC1. Bivalent rabbit anti-DNP antibody with the same association constant precipitated in physiological salt [3]. It has also been proposed [4] that chicken antibody precipitated in high salt with multivalent antigens because of salt induced aggregation. Hersh and Benedict[4] calculated S20,w of 14.0 and an apparent molecular weight of 550,000 for chicken IgG in 1.5 M NaCI. Aggregation of chicken IgG was verified by Van Orden and Treffers [5] and Kubo and Benedict [6] with molecular sieve chromatography. The main hypothesis resulting from aggregation studies has been that at physiological ionic strength chicken antibody does not precipitate with homologous antigen because of the inability to form lattices. Thus, aggregation of 2 or more antibody molecules creates a 'pseudo' multivalent antibody complex capable of lattice formation with antigen. Results of ligand binding studies and quantitative precipitin tests [3] indicated that the anomalous NaCI requirement of chicken antibody relates to the secondary phase of precipitation and not with primary antigen-antibody binding. On this basis a model was proposed [3] which involved a salt mediated conformational change of the chicken antibody molecule which permitted lattice formation due to induced polar orientation of the active sites. *This investigation was supported by Grant No. AI-08288 from the National Institutes of Health, Public Health Service. 771

772

JOAN S. GALLAGHER and E. W. VOSS, Jr.

To differentiate between aggregation or a conformational reorientation of the active sites of the molecule as a mechanism for the salt requirement in the chicken immune system, a condition other than high salt concentration would have to be found which would permit immune precipitation without aggregation of antibody. In this study chicken anti-DNP antibody, specifically purified by an immunoadsorbent, was precipitated by homologous antigen at low pH without aggregation. Binding properties of the antibody at low pH were investigated by quantitative precipitin tests, equilibrium dialysis and fluorescence quenching. MATERIALS AND METHODS

Source and preparation of chicken IgG antibodies A pool of hyperimmune sera from fifty chickens (White Rock) immunized intramusculary with 5.0 mg of DNP51BGG in complete Freunds adjuvant as previously reported [7] served as the source of antibodies. Chicken IgG antibodies were purified from this pool by immunoadsorption and IgG antibodies were resolved from IgM antibodies on DEAE cellulose in 0.05M potassium phosphate (PO,) buffer, pH 8.0.

Preparation of 125Ilabeledproteins Chicken, human and rabbit IgG were labeled with 125I (Na12SI, Iso/Serve Division, Cambridge Nuclear Corp.) by the ICI method[8] designed for 10 I groups to be substituted per mole of protein. Unreacted excess ~25I was removed by extensive dialysis against 0.05 M PO4, pH 8.0. Labeled products were greater than 95 per cent precipitable by 5 per cent trichloroacetic acid.

Precipitin analysis 2,4-Dinitrophenylated rabbit gamma globulin (DNP13RGG) prepared as described by Eisen [9] was used as the standard antigen in quantitative precipitin tests at low pH. Low DNP substitution was necessary in order to prevent nonspecific precipitation of antigen at low pH values. Precipitin assays were performed with 300/zg of antibody in a total volume of 2.0 ml in 0"05 M citrate buffer at 0.5 increments of pH ranging from pH 4"5 to 6"0. Reaction mixtures were incubated at 37° for 30 min before storing at 4 ° for 24 hr. Specific precipitates were collected by centrifugation, washed twice at 4° in 2 ml of 0.05 M citrate buffer at the appropriate pH. Washed precipitates were dissolved in 0.1 ml of 0.1 N NaOH before adjusting volumes to 3.0 ml with 0.05 M PO,, pH 8.0. Antibody and antigen concentrations were determined by absorbancy at 278 m/z and 360 m/z, respectively. An Elcm (278 m/z) for antibody of 15.1 [7] was used for quantitative determination of antibody. In the determination of antibody concentration in immune precipitates, the absorbance at 278 m/z was corrected for antigen contribution by using the A3n0/A27s ratio of 0"70 (DNPI3RGG).

Ultracentrifugation Analytical ultracentrifugation was performed with a Spinco Model E ultracentrifuge equipped with interference optics and a Beckman adjustable mono-

Precipitating Chicken Antibody at Low pH

773

chromometer for high intensity light (ultra-violet optics) and a photoelectric scanner. Interference optics were utilized to determine molecular weights by the Yphantis sedimentation equilibrium method[10]. Sedimentation values S20,w were determined utilizing ultraviolet optics. Densities of high salt solutions were obtained by interpolation of data from the International Critical Tables [11]. A Gaertner Microcomparator was used for analysis of photographic plates.

Molecular sieve chromatography Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) in 0.05 M sodium acetate (NaAc), pH 5.0 was packed to a bed volume of 1.9 × 76 cm. Fractions (0.5 ml) were collected at a flow rate of 0.4 ml/min and assayed for protein content by absorption at 278 m/z in a Beckman DU spectrophotometer.

Isoelectricfocusing Isoelectric focusing was performed using a l l 0 m l electrolysis column (LKB Instruments, Rockville, Md.) as described by Vesterberg et al.[12], p H gradients in 0-46 per cent sucrose of 3-10 were electrofocused for 72 hr at 300 V and pH 5-8 were electrofocused at 600 V for 72 hr. Position of 125I labeled proteins in the gradient was monitored by a RIDL gamma counter, pH was measured on a Metrohm E300 pH meter.

Sucrose density centrifugation To simulate electrofocusing conditions a 0-46 per cent sucrose gradient was made in 1 per cent ampholine solution pH 3-10 or 1 per cent e-Amino-NCaproic acid pH 6.6. Catalase (Worthington Biochemical, Freehold, New Jersey) used as a molecular weight marker, was assayed by decrease in absorption of hydrogen peroxide (H202) at 240 m/z[13]. Iodinated protein (0.1 ml of 500/zg/ml solution with specific activity 4 × 10 cpm//zg and 0.2 mg (0.05 ml of 4/zg/ml solution) catalase were layered on the gradient. Centrifugation was performed in a Beckman Spinco Model L centrifuge with a SW-39 rotor at 35,000 for 16 hr. Two tenths to 0"25 ml fractions were collected and monitored for catalase activity and radioactivity.

Immunodiffusion A microscope slide modification of Ouchterlony[14] double gel diffusion technique was used to assay for chicken IgG in electro-focusing fractions. Slides were coated with 1 per cent agar (Ionagar No. 2, Colab Laboratories, Inc., Chicago Heights, Illinois) in barbital buffer pH 8"6, ~ = 0"05. Rabbit anti-chicken plasma sera was placed in the central trough to develop precipitin bands.

Equilibrium dialysis Fifty microliter samples of purified antibody were dialyzed against 50/zl of various concentrations of 3H-e-DNP-L-lysine, using plexiglass chambers as previously described[3]. Antibody and ligand were dissolved in 0.05M citrate pH 5.0. Chambers achieved concentration equilibrium by standing for 20-24 hr at 4-5 °. Aliquots (25/~g) were removed (Drummond microliter pipettes) from each side of the chamber and dispersed into 10-0 ml of Bray's solution

774

JOAN S. GALLAGHER and E. W. VOSS, Jr.

[15]. Samples were counted in a Nuclear Chicago liquid scintillation spectrometer. Calculations of average intrinsic association constant (K0) were derived from a Scatchard plot[16] and the heterogeneity index (a) from a Sips plot as described by Eisen [17].

Fluorescencequenching Fluorometric titrations measuring binding of e-DNP-L-lysine (i.e. fluorescence quenching) by purified antibodies were performed as previously described [3] in an Aminco-Bowman spectrophotofluorometer according to the method of Eisen[18]. One ml samples of antibody (50/~g/ml) in 0-05M NaAc buffer, pH 5.1 were titrated in increments of 0.01 ml to 0"2 ml ligand (7.15 rap. mole/ml) in 0.05 M NaAc buffer pH 5.1. Protein fluorescence was excited with incident light at 300 mtx and emission measured at 350 mtz. Before adding hapten, 5-10 min were allowed for temperature (20-25 °) equilibration of the antibody solution to obtain stable fluorescence. Fluorescence values are expressed as a fraction of the initial fluorescence (i.e. before addition of hapten after corrections for solvent blank and for dilution owing to the volume of the hapten solution added). RESULTS

Precipitin analysis Figure 1 shows typical quantitative precipitin tests with 150/xg/ml of purified chicken anti-DNP antibody at various pH values. Maximum precipitation (89 per cent of antibody) was achieved at pH 5.0. Increasing the pH from 5"0 to 5.5 decreased immune precipitation of antibody by 76"5 per cent. From pH 5.5 to pH 6-0 antibody precipitation was further decreased (6.5 per cent). Thus, there was a total decrease in antibody precipitation of 83 per cent by raising the pH one unit. Lowering the pH from 5.0 to 4.5 decreased antibody precipitation 11.2 per cent to 78.6 per cent. Control studies showed that non-specific precipitation of chicken antibody or chicken normal IgG (NGG) did not occur in the pH range of 6.0 to 4.5. However, some non-specific precipitation of the antigen (DNP13RGG) occurred at pH 4.5, 5.0 and 5.5. At pH 4"5 and 5.0 approximately 80 per cent of the antigen added remained in solution, while at pH 5.5, 95 per cent of the antigen remained soluble. Control studies at low pH with antigen and chicken NGG (300/.~g/ml) resulted in the same percentages. In quantitative precipitin studies corrections were made for non-specific precipitation of antigen at each pH studied. Since there is a 30 fold variation in ionic strength over the pH range studied (from 0.100 at pH 4.5 to 0.300 at pH 8.0) controls were designed to determine whether ionic strength had any effect on immune precipitation at low pH. Quantitative precipitin tests at pH 5.0 (point of maximum immune precipitation) in 0"05 M citrate raised to an ionic strength of 0-300 with NaC1 in order to equal the ionic strength of 0.05 M PO4, pH 8.0, gave the same degree of precipitation as in 0.05 M citrate pH 5.0 (/x = 0.225). Lowering the ionic strength of the phosphate buffer at pH 8-0 to 0.225/z (0.0375 M PO4) did not result in immune precipitation of chicken antibody. Therefore, these results indicate that the low pH and not low ionic strength increased immune precipitation. These data

775

Precipitating Chicken Antibody at Low pH

i,oo

300[ t

190

80

70

60

~

50 ~

150

.,,j

30

75 20

0 ~

20

40

60

80

/~g DNPI3RGG

I00

120

....I 0

140

Fig. l. Results of quantative precipitin tests with 150/zg/ml of purified antiDNP antibody and DNP~aRGG at 0"5 pH increment increases from pH 4-5 to pH 8.0. Purified chicken anti-DNP antibody: pH 4.5°, pH 5"0Q, pH 5"5 ~, pH 6-0 A, pH 8.0 (1.5M NaCI)-.-. Control purified rabbit anti-DNP pH 5-0 I--q. Precipitin assays were performed in a total volume of 2"0 ml in 0.05M citrate buffer at the designated pH. Salt control was performed in • 0"05M PO4, pH 8.0. are consistent with the work of Goodman et al. [19] which showed a 60 per cent increase in precipitation of chicken antibody at pH 5.8 as compared to pH 7.7 in phosphate buffer at an ionic strength of 1.5. Control quantitative precipitin analyses of purified rabbit anti-DNP antibody at pH 5.0 in 0-05 M citrate and chicken anti-DNP antibody at pH 8.0 in 1.5 M PO4 were performed. Rabbit anti-DNP and chicken anti-DNP antibody gave similar quantitative precipitin curves with DNP~3RGG at pH 5.0 (Fig. 1). Nonspecific precipitation of rabbit antibody did not occur at pH 5-0. Quantitative precipitin curve of chicken anti-DNP at pH 8"0 in 1"5 M NaCI is shown in Fig. 1. Table 1 lists antibody/antigen (Ab/Ag) molar ratios at maximum antibody precipitation and after extrapolation to extreme antibody excess based on Fig. 1. No true equivalence zone (100 per cent precipitation of antibody and antigen) was evident in the low pH studies. At the various pH values studied both antibody and antigen were measured in the supernatant fluid. Thus, in the determination of molar ratios the amount of antigen in the precipitates was quantitatively determined instead of assuming that in the zone of antibody excess

"Fable 1. Molecular composition of i m m u n e precipitates A m o u n t s precipitated at zone of m a x i m u m precipitation

Molecular ratio of antibody to antigen3

© Z

Anti-DNP antibody Chicken z

Rabbits

Buffer 0-05 M citrate 0-05 M citrate 0-05 M citrate 0-05 M citrate 0-05 M PO 4 1.5 M NaC1 in 0"05 M PO4

pH

Ionic strength (tz)

Per cenO antibody

(/zg) Antibody

4.5 5.0 5.5 6.0 8.0 8.0

0-100 0.225 0-234 0.256 0.300 1.800

78.5 88-5 21-4 14.4 0 65.0

236.0 266-0 48-5 43-2 0 195-0

29-6 29-6 16-2 5-8 0 80.5

5.0

0.225

86-0

258.0

1See Fig. 1. ZAntibody concentration = 150/zg/ml; K0 = 1"7 × 10e M -1. aAntigen (DNPt3RGG; mol. wt. = 155,000). 4Obtained by extrapolation to zero antigen concentration. 5Assume mol. wt. = 155,00017]; K0 = 1-4 X 10aM -~. 6Amount antigen added.

Maximum precipitation zone

Extreme antibody excess zone 4

(80) 6 (100) (100) (100) (100) (100)

7.6 8.7 11-7 7"1

10-4 11-8 14-9 7"9

~,

2-2

5"9

¢L

20.0 (100)

10"4

12"0

(/zg) Antigen

©

©

777

Precipitating Chicken Antibody at Low pH

all antigen was precipitated. The molecular ratio of antibody to antigen in the extreme antibody excess zone at pH 5.0 for chicken anti-DNP antibody is 11"8, compared to 12.0 for rabbit anti-DNP. However, the Ab/Ag molar ratio in the extreme antibody excess zone for immune precipitation of chicken antiDNP in 1.5 M NaCI, pH 8"0 is 5"9; approximately half that of chicken and rabbit anti-DNP at pH 5"0. I m m u n e precipitates in the chicken anti-DNP system contained approximately three times more antigen at the point of maximum precipitation in 1-5 M NaCI, 0"05 M PO4, pH 8.0 than the immune precipitates in the zone of maximum precipitation at pH 5"0 (Table 1).

Ultracentrifug ation Analysis of chicken anti-DNP antibody by the Yphantis method of sedimentation equilibrium at pH 5.0 gave a molecular weight of 172,600 similar to the molecular weight of 178,00017] previously reported for chicken anti-DNP at pH 8"0. A plot of the logarithm of the concentration of protein vs. the radial distance squared from the center of rotation in 0.1 M NaAc, pH 5.0 and 0.05 M PO4, pH 8"0 gave a straight line indicative of a homogeneous protein in relation to molecular size. Table 2 shows that the molecular weight of chicken antibody in 0.1 M NaAc, pH 5.0 is independent of protein concentration as well as rotor speed. At a protein concentration of 0-53 mg/ml, chicken anti-DNP antibody had a S20,wof 7.30 in 0.05 M PO4, pH 8"0 and 6"75 in 0" 1 M NaAc, pH 5"0. Table 2. Weight average molecular weights of anti-DNP antibody I nitial concentration

Anti-DNP antibody Chicken

Rabbit

Solvent

pH

0"1 M NaAc

5"0

1.5 M NaCI*

8"0

2"0 M NaCI*

8"0

3.0M NaCI* 0"1 M NaAc 1.5 M NaCI*

8"0 5"0 8.0

Speed (rev/min) 17,980 21,740 12,590 15,220 12,590 15,220 12,590 17,980 15,220

(mg/ml) 0"25 0-50 171,786 176,728 ------

172,600 172 666 216000 200 000 347 000 283 581 390 000 155 700 157 592

*Stated NaC1 concentration plus 0.05 M phosphate buffer. Sedimentation equilibrium studies of chicken anti-DNP at Optimum NaCI Molarity*[3], 2.0M NaC1 in 0.05 M PO4, pH 8.0 at protein concentrations of 0"25 mg/ml and 0"50 mg/ml and at various rotor speeds indicated size heterogeneity because of the apparent dependence of molecular weight on rotor speed (Table 2). A Yphantis plot showed an upward curvature at the furthest distance from the center of rotation, suggesting a heterogeneous population of molecules relative to size. Molecular weight of chicken anti-DNP antibody in *Optimum NaC1 Molarity is the NaC1 concentration relative to protein concentration which will give maximum immune precipitation (i.e. 100 per cent antibody and antigen).

778

JOAN S. GALLAGHER and E. W. VOSS, Jr.

3"0 M NaCI 0.05 M PO4, pH 8"0, where only 50 per cent [3] immune precipitation of antibody protein occurred, was calculated to be 390,000 for a 0"50 mg/ml solution at 12,590 rev/min (Table 2).

Molecular sieve chromatography Gel filtration of chicken anti-DNP on Sephadex G-200 in 0"05 M NaAc, pH 5.0 resulted in the elution of one symmetrical peak of antibody protein from the column. Chicken anti-DNP eluted after Dextran Blue from the G-200 column was coincident with a 7S marker (purified rabbit IgG anti-DNP antibody). Precipitin analysis of the chromatographed chicken antibody revealed a salt requirement at pH 8.0 and no salt requirement at pH 5.0 for immune precipitation. Fractions analyzed directly from the column in 0.05M NaAc pH 5.0 precipitated with homologous antigen at physiological ionic strength. Aliquots from the column dialyzed to 0-05 M PO4, pH 8"0 required high concentrations of NaCI for immune precipitation. Fluorescence quenching titrations were preformed on chromatographed chicken anti-DNP, eluates from the column (0.05M NaAc, pH 5.0 buffer) quenched 41 per cent. After extensively dialyzing the fractions with 0.05M PO4, pH 8.0 the antibody's tryptophan fluorescence was quenched 43 per cent by bound ligand (~-DNP-L-lysine), compared to 41 per Cent quenching of the antibody's tryptophan fluorescence before chromatography.

Determination of isoelectricpoint Isoelectric focusing was used for determination of the pI of chicken anti-DNP and appropriate control proteins. Figure 2(a) shows the distribution of purified chicken anti-DNP antibody in a pH 3-10 gradient. The greatest proportion of the protein had a pI of 6.6 while a small fraction of protein had a pI of 5.6. The ratio of protein at pI 6.6 to pI 5.6 was approximately 3 : 1. The isoelectric profile for chicken NGG (Fig. 3(a)) is almost coincident with that of chicken antibody as shown in Fig. 2(a). Subsequent experiments in pH 5-8 gradients and with reversed polarity gave the same isoelectric profiles. Iodinated protein (125I) was used in isoelectric focusing for two reasons: (1) absorbance of ampholyte-sucrose solutions in the ultra-violet region masked the A278 absorbance of the protein, and (2) initial experiments indicated that high concentrations of chicken IgG (in excess of 8000 gg) were insoluble at their isoelectric point. Identical isoelectric profiles and pI values were obtained with the iodinated protein over a 100 fold protein concentration range (80-8000/~g). Therefore, it was concluded that protein concentration had no effect on the isoelectric measurements. Control experiments with low concentrations of unlabelled normal chicken IgG gave isoelectric profiles similar to those of 125I chicken IgG. However, the optical density profiles were not as satisfactory as the radioactive profiles. Thus, iodination of chicken IgG did not effect its isoelectric point. Identical specific activities cpm/A2~8 were calculated for the protein with a pI of 6.6 and protein with a pI of 5.6 indicating that the chicken anti-DNP antibody molecules were uniformly labelled with 125I. Immunodiffusion assay of fractions from electrofocusing experiments with rabbit-anti chicken plasma gave precipitin lines coincident with the pI 6.6 and

Precipitating Chicken Antibody at Low pH

779

35

I0

a

9

8

2O

7

15

6

~,o

5

4

(+~ I

O I "

I

I0

I

20

I

30

~

40

I

50

I

60

I

70

~o

;o

,oo I0

b

9

~'°

8

7

:z: 6

5 5

4

--) 01

(+;

,0

20

4 6 % SUCROSE

3o

I

4o

5o

6o

mt E F F L U E N T

70

~o 0%

;o

,0o

SUCROSE

Fig. 2. Isoelectric profiles of purified chicken anti-DNP antibody in pH 3-10 gradient at 300 V for 72 hr. (a) 100/~g of purified 12~I chicken anti-DNP antibody (b) fraction of 125I-chicken anti-DNP antibody (20 p.g) which traveled with the catalase marker in sucrose density gradient. 125Iprotein O, pH O.

5.6 protein peaks. No qualitative differences were noted in the extent to which the precipitin lines developed with each peak and they formed a line of identity. Determination of per cent activity of the two protein peaks obtained from the electrofocusing column were tested by readsorbability to the immunoadsorbent as previously described [7]. Ninety-eight per cent of the protein with a pI of 6.6 was readsorbed and 95-5 per cent of the protein with a pI of 5.6 was readsorbed. Test for free radioactivity (counts) in the pI 6-6 and 5-6 protein peaks showed that all counts were 100 per cent TCA precipitable. These results indicated that the protein was neither denatured nor 125I dissociated during the

780

JOAN S. GALLAGHER and E. W. VOSS, Jr. tO

62 ° 8

~20

~,0 5

4 I+)

o

" ,o"

2o

3o

,o

50

6o

7o

do

;o

,oo

~ to

~

4 ) I0

20

30

40

50

60

70

80

90

I00 I0

Nl(~

s

~5 ~

~

5

+)

4 6 % SUCROSE

--

rnl EFFLUENT

0 % SUCROSE

Fig. 3. Isoelectric profiles of control gamma globulins in pH 3-10 gradient at 300 V for 72 hr. (a) normal control, 2"4 mg chicken 125I IgG, (b) 125I rabbit anti-DNP antibody control, 3-40 mg, (c) heterogeneity control, 200/.~g of ~25Ihuman myeloma IgG. ~25Iprotein 0 , pH O. experiment and the two isoelectric points characterized biologically active proteins. A rabbit anti-DNP antibody control (Fig. 3(b)) gave an isoelectric point for the greater percentage o f protein at 5.4 in a pH 3-10 gradient. This value

781

Precipitating Chicken Antibody at Low pH

verified previous pI values reported for rabbit IgG [20]. It should be noted that the rabbit anti-DNP appeared more heterogeneous than chicken anti-DNP. Isoelectric focusing of heterogeneous normal human IgG gave an isoelectric profile for the protein encompassing three pI values (7.5, 7.7, and 7"9). Figure 3(c) shows the isoelectric profile for a human IgG myeloma protein. All of the protein electrofocused as a single peak indicative of a homogeneous pI 7.7 molecule. Comparison of the human myeloma to the heterogeneous chicken anti-DNP implies pI values of induced antibody are only average values for the population. Since the experimental pI determined for chicken anti-DNP antibody of 6"6 did not verify the pI of 5.3 for chicken IgG reported by Tenenhouse and Deutsch [21], sucrose density gradient centrifugation was performed in order to determine whether a monomer, dimer or other polymeric species was being electrofocused. Iodinated chicken anti-DNP antibody centrifuged at 35,000 rev/min for 16 hr in a 0-46 per cent sucrose gradient in 1 per cent ampholine solution pH 3-10 or in 1 per cent ~-Amino-N-Caproic acid (to simulate ampholytes) pH 6.7 traveled almost coincident with a 7S ~25I H u m a n IgG myeloma molecular weight marker and 0.91 ml behind the 250,000 mol. wt. marker catalase (Fig. 4). Approximately 20 per cent of the 12~Ichicken anti-DNP traveled with the catalase marker. Fractions containing the heavier chicken antibody were pooled and electrofocused in a pH 3-10 gradient. Figure 2(b) shows the

.°t .,q

E 20

/D~

.-? "6 "5

I0

\

.4

///rl,'

J

'3 ,2

.Y C

cl

e~

~q

,I ~1

i

5

46% SUCROSE

u

I0

15

FRACTION NUMBER

20

0

O%SUCROSE

Fig. 4. Sucrose density gradient profile of purified 1251chicken anti-DNP antibody C) with 125Ihuman IgG myeloma (S)and catalase [] markers. Experiment was performed in a 0-46 per cent sucrose gradient with 1 per cent pH 3-10 ampholine solution to simulate conditions of electrofocusing experiments. Centrifugation was performed in a Beckman Spinco Model L centrifuge with a SW-39 rotor at 35,000 for 16 hr.

IMMUNO. 7 / 9 - C

782

JOAN S. GALLAGHER and E. W. VOSS, Jr.

isoelectric profile from this experiment indicating a shift in the ratio of protein at pI 6.6 to pI 5.6 from 3 : 1 to 1 : 1. Determination of intrinsic association constants and valence

The average intrinsic association constant of chicken anti-DNP measured in 0-05 M citrate buffer pH 5.0 was 1.7 x 106M -1. This K0 value was essentially the same, within experimental error, as the values [3] reported for chicken antiDNP in PO4 buffer, pH 8.0 of 1.7 × 10°M -1 and at Optimum NaCI Molarity, 1"45M NaCI[3] of 1"8 x 106M -1. Heterogeneity index (a) of chicken anti-DNP was 0-87 at pH 5.0 in 0.05 M citrate. This is significantly higher when compared to 0.57 in 0.05 M PO4 buffer, pH 8.0 and 0.48 M NaCI, 0.05 M PO4, pH 8.0. The number of ligand binding sites per antibody molecule based on the experimentally determined molecular weight of 172,600 was 2.0 at pH 5.0 as shown in Fig. 5. This valence corresponds to the valence of 2 calculated at pH 8.0 and at Optimum NaCI Molarity [3]. Average intrinsic association constant of a control, rabbit anti-DNP antibody at pH 5"0 in 0.05 M citrate buffer was 1.4 x 106 M -1 and the measured valence was 2.0. These results are comparable to a valence of 2.0 and K0 of 1.4 X 10nM -1 measured at pH 8.0 and in 1.45M NaCI. The heterogeneity index of rabbit anti-DNP antibody, at pH 5.0, was also significantly higher (0.81) when compared to the heterogeneity index in pH 8.0 and in 1.45 M NaC1 (0.39). DISCUSSION Precipitation o f chicken anti-DNP antibody with homologous antigen at low pH and low ionic strength has enabled the study of immune precipitation in the chicken system in the absence of high concentrations of NaC1. Immune precipitation of chicken anti-DNP antibody with homologous antigen at physiological salt concentrations was enhanced by raising the proton concentrations 1000 fold to a pH of 5.0. Precipitin curve shown in Fig. 1 is similar to precipitin curves obtained with the chicken anti-DNP system in high concentrations of salt[3]. However, in contrast to high salt precipitin studies[2, 3] Ab/Ag molar ratios extrapolated to extreme antibody excess at pH 5.0 give ratios similar to bivalent rabbit anti-DNP antibody (11.8 vs. 12.0 respectively). These ratios suggest that the two precipitating antibodies (similar K0) at low pH saturate the multivalent antigen with a similar number of antibody molecules. Chicken antiDNP antibody in 1.5 M NaC1 at pH 8"0 has a molar ratio of Ab/Ag in extreme antibody excess zone of 5.9 (Table 1) or half that of the bivalent rabbit reference. This difference in the molar ratio is probably attributed to the fact that approximately 2-3x more antigen was found in the immune precipitates with the high salt system than with the low pH system. The lower ratio is probably a reflection of this additional precipitation of antigen. There are two possible reasons for this higher amount of antigen precipitated, (1) antigen is trapped by the aggregation of chicken antibody molecules or, (2) antigen is aggregated by 1-5 M NaC1. The latter proposal is supported by the observation that non-specific precipitation of antigen begins at 2"0 M NaCI [3]. Molecular weight of chicken anti-DNP antibody at pH 5"0, determined by Yphantis sedimentation equilibrium was 172,600 comparable to 178,000 at pH

Precipitating Chicken Antibody at Low pH

783

.8 -6

4-0

~-4

Q

\.2 t.. 0 G ~4 I 4

3"0

log C i

I'0

I

I -o

2-o r

Fig. 5. Scatchard and Sips plot of binding of e-DNP-L-lysine by chicken anti-DNP from equilibrium dialysis data at pH 5-0. Fifty/zl samples of purified antibody were dialyzed against 50tzg of various concentrations of aH-~-DNP-L-lysine using plexiglass chambers. Antibody and ligand were dissolved in 0.05 M citrate buffer, pH 5-0. Chambers achieved concentration equilibrium by standing for 20-24 hr at 4-5 °. In all cases r is moles hapten bound/mole antibody, c is free hapten concentration M/L and n is the limiting value for r at infinite C. 8"0[7]. Hersh et al. [22] reported a molecular weight for chicken IgG in 0.1 M NaCI pH 5.0 of 170,000. I m m u n e precipitation by the monomeric form of chicken anti-DNP at pH 5.0 in physiological salt shows that antibody aggregation is not necessary for precipitation of antibody by antigen in the chicken system. In an attempt to determine whether aggregation of the chicken IgG molecule was the single factor causing immune precipitation of chicken antibody in high salt, molecular weight of purified chicken anti-DNP was determined in 2.0M and 3.0M NaC1. Previous studies[3] showed that while maximum immune precipitation occurred at 2.0M NaCI, immune precipitation was decreased 50 per cent in 3.0 M NaCI. This decrease in precipitation can not be attributed to a shift in equilibrium as previously proposed [23] since binding data illustrate the stability of the chicken antibody combining site to relatively extreme conditions. At high concentrations of salt (0.5-3.0M NaCI) and low pH 0.05M citrate pH 5.0 and pH 4-5) the average intrinsic association constant of chicken

784

JOAN S. GALLAGHER and E. W. VOSS, Jr.

anti-DNP antibody (1.7 × 106M -~) and valence (2.0) was the same as measured at physiological conditions in 0.05M P04, pH 8.0. The K0 (1.4× 106M -1) and valence (2.0) of rabbit anti-DNP antibody were identical in all environments tested; 0.05 M PO4, pH 8.0, 1.45 M NaC1 in 0.05 M PO4, pH 8.0 and 0.05 M citrate pH 5.0 despite the fact that high concentrations of salt inhibit precipitation in the rabbit system. If aggregation mediates immune precipitation of chicken antibody then one would predict decreased aggregation of the chicken IgG molecule at 3.0 M NaCI to coincide with decreased immune precipitation at this point. Sedimentation equilibrium studies of chicken anti-DNP antibody in 2.0 M NaC1 (Optimum NaC1 Molarity) and 3.0 M NaC1 at protein concentrations of 0"5 mg/ml and a rotor speeds of 12,590 rev/min, suggested dimers 347,000 and 390,000 respectively (Table 2), as the major molecular weight species in a heterogeneous population of molecules. The fact that the molecular weight in 2"0 M NaCI and 3"0 M NaC1 is essentially the same while there is a 50 per cent decrease in immune precipitation indicates that aggregation is not the unique mechanism for the salt requirement in the chicken immune system. Because of the antibody's increased ability for immune precipitation at low pH it was important to determine the relationship of the point of maximum immune precipitation (high proton concentration) to the pI of the protein. The isoelectric point of chicken IgG experimentally determined by electrofocusing was 6"6. This was 1.6 pH units higher than the pH at the point of maximum immune precipitation of 5.0 and 1.3 pH units higher than the values of 5.3 reported by Tenenhouse and Deutsch [21] from moving boundary electrophoresis studies. Control electrofocusing experiments with rabbit anti-DNP antibody and human normal IgG verified previously determined [20] isoelectric ranges. Possibly, the discrepancy between these values (6.6 vs. 5.3) can be attributed to two factors. First, the same molecular form was not being electrophoresed (i.e. electrophoresis of a monomer vs a dimer or other polymeric form). Results of sucrose density centrifugation experiments which simulated conditions of electrofocusing showed that the pI of 6.6 characterized the monomeric unit of chicken IgG. It was noted that 20 per cent of the protein was almost coincident with the 250,000 mol. wt. marker. Pooling and electrofocusing this material enriched for the pI 5"6 species suggesting that a fraction of chicken IgG heavier than a monomer would have a lower pI than 6.6. Second, Tenenhouse and Deutsch [21] determined the isoelectric point at an ionic strength of 0.1. Electrofocusing experiments reported above were performed at an ionic strength of 0"01 (possibly lower) therefore approximating isoionic conditions. The isoionic point of protein including globulins are generally higher than the isoelectric point near 0.1 ionic strength [24]. Previous work[3] has shown that the anomalous NaCI requirement for chicken antibody relates to the secondary phase of precipitation and not with primary antigen-antibody binding. Since molar ratios of Ab/Ag in zone of antibody excess from low pH-studies suggest that chicken antibody acts like the bivalent rabbit antibody control, it is proposed that studies to elucidate the mechanism for immune precipitation of chicken antibody be conducted at experimental conditions of low pH and low ionic strength. Possibly high propon concentration, like high salt, induces a conformational reorientation

Precipitating Chicken Antibody at Low pH

785

o f the chicken I g G molecule by the previously p r o p o s e d m o d e l [3]. T h i s m o d e l could be e x p e r i m e n t a l l y tested at low p H a n d low ionic s t r e n g t h in the absence o f a g g r e g a t i o n o f chicken IgG. REFERENCES Orlans E., Rose M. E. and MarrackJ. R., Immunology 4, 262 (1961). BanovitzJ., Singer S.J. and Wolfe H. R.,J. Immun. 82,481 (1959). Gallagher J. S. and Voss E. W., Immunochemistry 6,573 (1969). Hersh R. T. and Benedict A., Biochim. biophys. Acta 115,242 (1965). Van Orden D. E. and Treffers H. P.,J. Immun. 100,659 (1965). Kubo R. T. and Benedict A. A.,J. Immun. 103, 1022 (1969). GallagherJ. S. and Voss E. W., Immunochemistry 6, 199 (1969). MaFarlane S. S., Nature, Lond. 182, 53 (1958). Eisen H. N., Methods in Medical Research (Edited by Eisen H. N.), Vol. 10, p. 94. Yearbook Medical Publishers, New York (1964). 10. Yphantis D. S., Biochemistry 3, 3 (1964). 11. International Critical Tables. National Research Council (Edited by Washburn E. W.), Vol. 3, p. 79. McGraw-Hill, New York (1928). 12. Vesterberg O., Wadstrom T., Vesterberg K., Svensson H. and Malmgren B., Biochim. biophys. Acta 133,435 (1967). 13. Beers R. F. and Sizer I. W.,J. biol. Chem. 195, 133 (1952). 14. Ouchterlony O., Actapath. microbiol, scand. 32,231 (1963). 15. Bray G. A., Analyt. Biochem. 1,279 (1960). 16. Schatchard G., Ann. N.Y. Acad. Sci. 51,660 (1949). 17. Eisen H. N., Methods in Medical Research (Edited by Eisen H. N.), Vol. 10, p. 106. Yearbook Medical Publishers, New York (1964). 18. Eisen H. N., Methods in Medical Research (Edited by Eisen H. N.), Vol. 10, p. 115. Yearbook Medical Publishers, New York (1964). 19. Goodman M., Wolfe H. R. and Goldberg R.,J. Immun. 72,440 (1954). 20. Kabat E. A. and Mayer M. M., Experimental Immunochemistry, p. 327. C. C. Thomas, Springfield, Ill. (1961). 21. Tenenhouse H. S. and Deutsch H. F., Immunochimistry 3, 11 (1966). 22. Hersh R. T., Kubo R. T., Leslie G. A. and Benedict A. A., Immunochemistry 6, 762 (1969). 23. Heidelberger M., Kendall F. E. and Teorell T.,J. exp. Med. 63,819 (1936). 24. Mahler H. R. and Cordes E. H., Biological Chemistry, p. 54. Harper and Row, New York (1966). 1. 2. 3. 4. 5. 6. 7. 8. 9.