Heterogeneity of 125I-labeled tetanus toxin in isoelectric focusing on polyacrylamide gel and polyacrylamide gel electrophoresis

ARCHIVES OF BIOCHEMISTRY AND BIOP~SICS

Vol. 200, No. 1, March, pp. 208-215, 1980

Heterogeneity of 1251-Labeled Tetanus Toxin on Polyacrylamide Gel and Polyacrylamide

in Isoelectric. Focusing Gel Electrophoresis

BIRGIT AN DER LAN,’ WILLIAM H. HABIG, M. CAROLYN HARDEGREE, AND ANDREAS CHRAMBACH* Bureau of Biologics, Division of Bacterial Products, Food and Drug Administration, *Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Beth,esda, Maryland 20205

and

Received July 24, 1979 Tetanus toxin isolated from Clostridium tetani and radioiodinated by both the HunterGreenwood and the Bolton-Hunter procedures was analyzed by isoelectric focusing on polyacrylamide gel and by “quantitative” gel electrophoresis. Immediately after iodination, the focused preparation exhibited a single protein component with a pZ’ of 5.1, while after storage it was progressively transformed into a protein with a pZ’ of 5.9. Formation of the second component could not be prevented by storage in liquid nitrogen (-196°C). Although polyacrylamide gel electrophoresis at either pH 3.7 or 11.0 did not reveal this heterogeneity, it provided physical parameters describing molecular size and net charge, by which the molecule can be recognized.

In recent years analytical polyacrylamide gel electrophoresis (PAGE)2 has been successfully applied to defining the physical properties of ligand-receptor complexes (e.g., Refs. (1, 2)). In preparation for a similar investigation designed to extend our present knowledge (3-7) of the molecular * Reprint requests should be sent to Birgit An der Lan, Bureau of Biologics, FDA, Bldg. 29, Room 412, 8800 Rockville Pike, Bethesda, Md. 20205. 2 Terms and abbreviations used: Bis, N,N’-methylenebisacrylamide; %C, percentage crosslinking of gels; DATD, N,N’-diallyltartardiamide; Ferguson plot, plot of log R, vs %T; IFPA, isoelectric focusing on polyacrylamide gel; Ks, retardation coefficient, slope of the Ferguson plot; M,, electrophoretic mobility in free solution; MZE, multiphasic zone electrophoresjs; PAGE, polyacrylamide gel electrophoresis; pl’, apparent isoelectric point; PI/LAMBDA boundary, moving boundary at the front of the operative resolving phase; I?, molecular geometric mean radius; RM units, electrophoretic mobility relative to sodium; SDS, sodium dodecyl sulfate; %T, total monomer concentration (g/lOOml); TEMED, N,N,N’,N’-tetramethylethylenediamine; TT, tetanus neurotoxin; TTi, component of iP51-labeled TT with pZ’ of 5.1; TT,, component of *Z51-labeled‘PI’ with a pZ’ of 5.9; V, valence (net H+/ molecule); Y,, antilog of the extrapolated y-intercept on the Ferguson plot. 0903-98fX/80/030206-10$02.00/O Copyright 0 1980 by AcademicPress, Inc. All rights of reproductionin any form reserved.

nature of the complex formed between tetanus toxin (TT) and its receptors, we undertook a detailed characterization of the electrophoretic properties of the labeled ligand. Previous physical characterization of lz51labeled TT had been based on gel filtration, in which the labeled toxin eluted close to immunoglobulin G (8) and lactate dehydrogenase (9), giving a rough estimate of molecular size which was in agreement with values reported for native TT (10). The homogeneity of iodinated TT had been demonstrated by PAGE in the presence of SDS (under oxidative conditions), where it exhibited a single zone (9, 11). The purposes of this study were, therefore, to define by more rigorous physical methods TI’ relative the size and charge of 1251-labeled to native TT, to investigate the homogeneity of the labeled product by methods previously applied to other proteins (12), to compare the bioactivity and electrophoretic behavior of toxin radioiodinated by two different procedures (13, 14), and to investigate the stability of. the iodinated toxin. Because of their resolving power, “quantitative” PAGE (15) and isoelectric focusing on polyacrylamide gel (IFPA) (16) were used for this investigation.

206

‘“I-TETANUS MATERIALS

TOXIN IN GEL ELECTROPHORESIS

AND METHODS

Tetanus Toxin and Other Proteins TT was purified from the Massachusetts C, strain of Clostridium tetani as described previously (4). Standard proteins for molecular weight estimations were bovine serum albumin, phosphorylase b, thyroglobulin type 1, ribonuclease A, and y-globulin (Cohn Fraction II) (Sigma), aldolase (Boehringer), ferritin (Schwa&Mann), myoglobin (Calbiochem), chymotrypsin and human hemoglobin (Worthington). Orosomucoid was a gift from Dr. N. Nwokoro (NICHD, NIH). (2)

Radioiodination

(a) By the Bolton-Huntermcedure (13). ‘IT, 100pg, was conjugated via its free amino groups with 1 mCi of an iodinated p-hydroxyphenylpropionic ester (New England Nuclear, 1500 Ci/mmol) to a final specific activity of 0.7 mCi/mg. (b) By tke Hunter-Greenwood procedure (14). TT, 100 pg, was labeled in its tyrosine residues with 500 j&i of carrier-free sodium [lz51]iodide (New England Nuclear) to a final specific activity of 0.7 mCi/mg. After iodination by either procedure the labeled protein was separated from the iodination mixture by gel filtration on a 0.85 x 23.0-cm column of Sephadex G100. To increase their stability the iodination products were stored in buffers containing extraneous protein: the Bolton-Hunter labeled toxin in 0.05 M phosphate buffer, pH 7.2, containing 0.25% gelatin and the Hunter-Greenwood labeled toxin in 0.02 M Tris/acetate, pH 7.2, containing 0.25% bovine serum albumin. (3)

Bioassay

Neurotoxic activity of labeled TT was determined by the mouse lethality test (4), using NIH general purpose mice weighing 15 to 18 g. One-tenth milliliter of each dilution (diluted in phosphate-buffered saline, pH 7.4, containing 0.2% gelatin) was injected subcutaneously into the right inguinal fold of two animals. The endpoint was based on the greatest dilution causing death of both mice in 96 h. Because the amounts of toxin recovered from PAGE gels were too small to estimate mouse lethal doses, unlabeled TT was measured by a different assay, based on a scale of l-5 describing the severity of the .symptoms 24 h after injection (5 = death, 4 = animal prostrate, 3 = paralysis of foot, 2 = marked scoliosis, 1 = mild seoliosis). The bioactivity of lZSI-labeled TT recovered from IFPA was assessed by observing death times of mice after injection.

(4) Optimization of the pH of PAGE The pH of PAGE was optimized using stacking gels only (15, 1’7,18). The multiphasic zone electrophoresis

207

(MZE) buffer systems (19-21) listed in Table I and a gel concentration of 5 %T, 15 %COATD (22) were used. Ionic strength (ZETA) was 0.01 M in all systems. In all cases the initiator concentrations were 0.015% potassium persulfate, 5 x 10B4%riboflavin, and 1 ~1 TEMED/ml gel in systems of negative polarity, and 2 ~1 TEMED/ml gel in systems of positive polarity [for procedure see Ref. (15)]. Gel tubes of 6 mm i.d. and of 18cm length, containing 3.5 ml gel, were employed in a six-chamber Pyrex apparatus (18). Current was regulated at 1 mA/tube or at 0.25 milltube for overnight runs. Stacks were marked by bromphenol-blue (negative polarity) or thionin (positive polarity), and were then excised and analyzed for radioactivity.

(5) Ferguson Plots and Physical Characterization in PAGE PAGE was carried out in MZE buffer systems 2950 and 35.2.V. as described (15). The stacking gel concentration was 3.125 %T, 20 %Cels; resolving gel concentrations ranged from 5 to 10 %T, 2 %Csis(negative polarity) and 3.4 to 8.8 %T, 2 %Csis (positive polarity). Tracking dyes for the PI/LAMBDA boundary were bromphenol blue (negative polarity) and methyl green (positive polarity). Ferguson plots were constructed, and their slopes (retardation coefficient K,) and y-intercepts ( YO)evaluated statistically (23, 24). By means of a standard curve KR values were translated to values of geometric mean radius, R, and of molecular weight, M, (23-25). The anionic protein standards used are listed in Table IIIA and the cationic standards in Table IIIB. Standard and unknown proteins were assumed to be globular (23,24). Y, values were translated to values of valence (net H+/molecule) and free mobility (M,) as described (23, 24). All computations used the “PAGE-PACK” programs of Rodbard (15). The surface charge density of standard proteins and 1251-labeledTT was calculated as the ratio of valence to molecular surface area [molecular surface area = (molecular weight) 213x 5.57 x 10m2nm*], and is expressed as H+ per square nanometer. (6)

Analysis of Labeled Contaminants of 12V-Labeled TT by PAGE

To determine whether the labeled material which stacked in system-2950 was homogeneous, or whether protein aggregates or small molecular weight contaminants migrated within the same mobility limits as TT, the electrophoretic behavior of 1251-labeledTT in a stacking gel alone (a) was compared with its behavior upon molecular sieving (b). Aliquots (30 ng) of lz51labeled TT were electrophoresed into either (a) stacking gels only (as described in section 4), or (b) through similar stacking gels into 4-ml resolving gels (12 %T, 2 %C&, both prepared in stacking gel buffer only. After PAGE the distribution of label in cases (a) and (b) was analyzed as follows: (a) the stacking gels

208

AN DER LAN ET AL.

were sectioned into gel surface (i), the gel between its surface and the stack (ii), the stack (iii), and the rest of the gel (ivt; (b) the composite gel containing both a stacking gel and a resolving gel was sectioned into stacking gel (v), resolving gel between its origin and the PI/LAMBDA boundary (vi), the PI/LAMBDA boundary marked by bromphenol blue (vii), and the rest of the gel (viii). The percentage of the total amount of label applied was calculated for each section.

(7~;&ectric

Focusing on Polyacrylamide

Gels, 6 %T, 15 %CuATD,1.5 ml/gel tube, were subjected to IFPA at 0-4°C and at a regulated potential of 40 V/cm. pH gradients were determined semiautomatically (26). Other conditions of IFPA are described (2’7, 28). (a) Development of suitable pH gradients forelectrofocusing of ~251-lubeledTT. Using a 1:l mixture of 2% Ampholine preparations (LKB) with pZ ranges of 4-6 and 6-8 (“narrow pZ range”) a series of catholytes with increasing pK(NH,) and a series of anolytes with decreasing pK(COOH) were applied with the purpose of forming a pH gradient, stable for at least 40-60 h, which was shallow around pH 5.1, the previously reported pZ of lT (29). A solution of 0.1 M imidazole proved suitable as catholyte, and 0.1 M acetic acid as anolyte (38). When experiments were aimed at focusing a component with a more alkaline pZ’ at the center, rather than at the alkaline end of the pH gradient, the alkaline range was extended by using 0.1 M arginine as catholyte and substituting the Ampholine preparation with a pZ range of 4-8 by one with a range of 3.5-10 (“wide pZ range”). (b) Isoelectric focusing of ‘Z51-kzbekdTT. ‘I7 (usually 60,000 to 100,000 cpm corresponding to about 30 ng of toxin) was applied cathodically in 0.02 to 0.05 I catho1yt.eand 30% sucrosetogether with 100M orosomucoid.3 After focusing and measuring the pH gradient, gels were sliced into l-mm slices (Hoefer Scientific Co., San Francisco, Calif. No. SL-280) and analyzed for radioactivity. (The procedure of gel slicing with this apparatus applicable to electrofocusing gels of 6 %T, 15 %CDATDwill be provided on request.) After adding 0.2 ml of 0.05 M KC1 the pH values of the slices containing the peaks of radioactivity were confirmed by manual pH measurement.

(8) El&ion of lz51-Labeled TT from Gel Slices TT was recovered from PAGE slices by allowing it to diffuse for a few hours at room temperature into 3 Extraneous protein is added to the load to saturate polyacrylamide adsorption sites (37). In view of its high acidity orosomucoid was selected so that it would sweep the entire gel when loaded cathodically.

0.2 ml of 0.05 M phosphate buffer, pH 7.2, containing 0.25% gelatin (the volume of each gel slice is about 0.028 ml). TT was eluted from IFPA slices overnight at 6”C, using otherwise similar conditions.

(9) Isotope analysis 125I was measured using either a Beckman or a Searle gamma spectrometer. RESULTS

(1) Bioactivity of Iodinated Preparations Immediately after iodination the specific activity of the toxin labeled by the BoltonHunter procedure was similar to that of native toxin (10’ mouse lethal doses/mg protein). However, the toxin labeled by the Hunter-Greenwood method exhibited onethird to one-fourth of the bioactivity of the toxin labeled by the other procedure. The toxicity of the Bolton-Hunter iodination product was measured at four different time intervals after iodination and was found to decrease fourfold during 9 weeks of storage. (2) Optimization of pH for PAGE Fractionation of Labeled Products of the Radioiodination of TT The lowest pH at which anionic labeled products of the iodination reaction of TT could be concentrated by stacking [which is, by definition, the optimum resolving pH for PAGE (15, 17, 18)], with recovery of 79%, was 9.6 (buffer system 2860). At pH 10.4 (buffer system 2950) the recovery of lz51Iabeled TT in the stack was 86% (Table IA). Cationic 1251-labeIedTT was stacked in an analogous manner using the buffer systems shown in Table IB. Maximum recoveries were of the order of 50%. The highest pH at which such recovery was obtained was 4.88 (Table IB). Negligible recovery at pH 3.16 probably reflects acid denaturation. (3) Ferguson plots of V-Labeled

TT

PAGE analysis of lz51-labeledTT was carried out at an operative resolving pH of 11.0 (buffer system 2950). This buffer system was selected becauseit gave the highest recovery of label in the stack [see section 21. Gel concentrations -_” -~ --~~~~ rantine m ‘2 from 5 to 10 %T. 2 %CRir

‘=I-TETANUS

209

TOXIN IN GEL ELECTROPHORESIS TABLE IA

RECOVERY IN THE STACK OF ANIONIC RADIOIODINATION PRODUCIV

System

1906

1954.2

1933.2

1935

2254

2325

2860

2950

RM(l,ZETA) RM(2,BETA)

0.052 1.280

0.025 0.249

0.049 1.280

0.093 1.280

0.045 1.280

0.068 0.592

0.096 0.789

0.064 1.280

pH (ZETA) Recovery (%)

6.77 12

6.43 16

7.10 16

7.42 14

7.81 18

8.07 13

9.63 79

10.45 86

L Time after iodination varied from 4 to 43 days, but did not correlate with percentage recovery in the stack.

were used (Fig. 1). The gel patterns and relative electrophoretic mobilities (R,) of TT labeled by either the Bolton-Hunter or the Hunter-Greenwood procedures were not significantly different. The Rfs of biological activity of unlabeled TT did not appear significantly distinct from those of the iodinated derivatives at the gel concentrations at which unlabeled TT was analyzed (6 and 9 %T) (Fig. 1). The R, values corresponding to the peak positions of both labeled TT preparations were plotted against gel concentration (Ferguson plot). The slope of this plot (K,J yields a measure of molecular size and the y-intercept (Y,) a measure of net charge. Ferguson plots for each preparation of iodinated TT, including two points for noniodinated TT, together with the joint 95% confidence envelopes of KR and YO,are shown in Fig. 2. Because these elliptical confidence envelopes for both species overlap, the two iodination products are physically indistinTABLE IB RECOVERY IN THE STACK OF CATIONIC RADIOIODINATION PRODUCTS”

System

7.1

18.2

6.15

35.2

RM(l,ZETA) RM(2,BETA)

0.042 1.490

0.036 0.258

0.025 0.219

0.035 0.215

pH (ZETA) Recovery (%)

3.16 1

3.67 57

3.84 58

4.88 50

a Stacking of TT at acid pH was investigated using toxin which had been stored for 30 days. The relatively low recovery in the stack (50% compared to 80% in alkali) may be in part accounted for by preferential or exclusive stacking of iodination products with pZ higher than that of TT,, i.e. 5.1.

guishable under the conditions of the analysis (24). Using a standard curve of KR as a function of molecular radius (R ) for standard globular proteins (Fig. 3), the values of molecular radius and weight were derived for TT iodinated by both procedures (Table II). Table II also provides the values of molecular net charge (valence, V) and free mobility (M,) computed on the basis of Y, and R for both labeled toxins. Tables IIIA and B show these parameters derived for the standard proteins used. The calculated charge densities for the anionic standard proteins lie between 6.7 and 32.0 x lo-*, and when plotted as a function of pl, are inversely related to pZ. The calculated charge density for anionic 1251-labeledTT of 3.6 x lo-* is considerably less than the value expected from its pl’ on the basis of such a plot. Ferguson plot analysis was also performed on 1251-labeledTT at an operative resolving gel pH of 3.7 (buffer system 35.2.V) in the same manner. Table II summarizes the relevant physical characteristics of labeled TT at acid pH. They are indistinguishable within 95% confidence limits from those obtained at pH 11. The ratios of V/molecular surface area for the cationic standards (between 12.6 and 15.5 x lo-*) are several-fold higher than that of cationic lz51-labeled TT (5.4 x lo-*). These standards are all basic proteins with pi’s at least two pH units higher than the measured pl’ of 1251-labeledTT. (4) Iodinated Contaminants of ‘*Y-Labeled TT Detected by PAGE and Their Removal by Gel Electrophoretic Methods

After separating bound from free iodine by gel filtration, 86% of freshly iodinated

210

AN DER LAN ET AL.

toxin stacked at pH 10.45 (segment iii). Approximately 10%of the label was retained at the surface of the stacking gel (segment i), and is presumed to represent an aggregated protein contaminant with a molecular weight in excess of 5 x lo5 (22). It is removed by retention at the surface of the stacking gel. The remaining percentage of the label migrated ahead of the stack (segment iv), thus separating it from stacked TT. A negligible amount of label was found in segment ii. To determine whether the labeled material migrating within the stack contained any other iodinated contaminants, the same amount of labeled protein as was applied to stacking gels alone was electrophoresed through the stacking gel into a restrictive resolving gel (12 %T, 2 %C,J, in which protein, including toxin, would unstack, while more rapidly migrating contaminants would remain stacked and thus move with the moving boundary front marked by tracking dye. Analysis of various segments for

FERGUSON

PLOTS OF 125l-lT

Rf

4

8 %T

12

oa8 0.10 0.12 KR

FIG. 2. Ferguson plots of TT iodinated by (a) the Bolton-Hunter or (b) the Hunter-Greenwood procedure. PAGE conditions as in Fig. 1. The dotted lines delineate 95% confidence envelopes. Solid lines were obtained by least squares linear regression analysis of the data points shown. For comparison the two points derived for uniodinated toxin are shown on both plots (open triangles). The joint 95% confidence envelopes of the slopes of these lines (retardation coefficients, K,) and their y-intercepts ( Yo), which are measures of molecular size and net charge, respectively, are shown in the right panels.

distribution of radioactivity showed that 10% of the label remained in the stacking gel (segment v, the same contaminant as found in segment i), while 85% migrated in the resolving gel behind the moving boundary (segment vi). The remaining 5% of the label was associated either with the moving boundary (segment vii), or was found beyond it (segment viii). This rapidly migrating FIG. 1. Gel patterns of anionic iodinated and native contaminant is presumably the same as that TT in PAGE at several gel concentrations: l”l’ iodinated in segment iii. The label in both segments vii by either the Hunter-Greenwood (solid lines) or the and viii could be removed completely by Bolton-Hunter (dashed lines) procedures was com- dialysis, demonstrating that it is of small pared and plotted as cpm/mm gel slice. PAGE (MZE molecular weight. The amount of label found buffer system 2950,0-4°C 2 %C,,,) was carried out at within the stack in the stacking gel corthe gel concentrations shown; about 70,000cpm of toxin responds to the amount which is unstacked was applied to each gel 14 days after iodination. The in the resolving gel (segment vi). Accorddotted lines refer to relative biological activity of the uniodinated toxin; about 200 mouse lethal doses ingly, the stack does not contain significant amounts of iodinated contaminants (1% or of native toxin were applied to each gel. The biological activity scale is defined under Materials and Methods. less).

Y-TETANUS

TOXIN

211

IN GEL ELECTROPHORESIS

Methods (data for Hunter-Greenwood procedure and for “narrow” pI range not shown).

I

1.0

2.0

I

3.0

4.0

5.0

nl

R lnm) FIG. 3. Standard curve for molecular weight determination of TT by PAGE: PAGE conditions as in Fig. 1. Standard proteins used to obtain the data points shown are listed in Table IIIA. Dotted and solid lines are defined as in Fig. 2. The type of standard curve shown is that for globular proteins; hydration is neglected; partial specific volumes are assumed to be 0.74 (21).

(5) Determination of Apparent pI (PI’) of 1251-Labeled TT by Electrofocusing Immediately after iodination by either procedure 1251-labeled TT was subjected to electrofocusing in the pI ranges 4-8 and 3.5- 10 for various time periods ranging from 3 to 40 h. Electrofocusing yielded a single major peak of radioactivity, designated as TT, (Fig. 4a). After 40 h of electrofocusing the peak of TT, approached a constant pH giving a PI’ of 5.1 (Fig. 4b). An indistinguishable pl’ value was obtained for the products of the Hunter-Greenwood and the Bolton-Hunter procedures using carrier ampholytes with either the “wide” or “narrow” pI ranges defined under Materials and TABLE

(6) Iodinated Component TT, Derived from ‘251-Labeled TT and Detected by Electrofocusing After use of either iodination procedure a second iodinated peak, designated as TT2, formed progressively with storage time (Fig. 5). Its pl’ was between 5.8 and 5.9 (Figs. 4b and 5a). The rate at which component TT, was generated was the same whether ‘Y-labeled TT was stored at - 14°C or at -196°C. The relative recovery of TT, in electrofocusing decreased in proportion to storage time after iodination, while the relative recovery of component TT, correspondingly increased (Fig. 5b shows this result with reference to the Bolton-Hunter product). Both TT, and TT2, when eluted from IFPA gel slices (see section 8), caused death in mice after similar time intervals, and were therefore approximately equally bioactive. Thus TT, appears to be formed from TT,. (7) Recovery of Labeled TT from Gel Slices After PAGE native toxin could be recovered by diffusion for a few hours. In IFPA 50% of the isoelectric labeled toxin was recovered by the same method (focusing load 30 ng or 3 pg per tube). However, bioactivity could only be recovered when the IFPA load was 3 pg per tube or more. DISCUSSION

Heterogeneity

of Iodinated

TT

In agreement with the data from gel filtration (9) and SDS-PAGE (11) of iodinated II

Iodination

PH

KR x 1oZ

UK, x 102

yo

UYO

R

M,

V

MO x 1O-5

Bolton-Hunter Hunter-Greenwood Bolton-Hunter

11.0 11.0 3.7

10.11 9.48 12.94

0.57 0.73 0.98

2.4 2.2 1.9

0.23 0.27 0.25

3.6 3.5 3.3

162,000 145,000 120,000

16.5 14.5 18.3

6.0 5.5 7.4

Abbreviations used: (T, standard deviation; R, molecular radius in nm; V, net H+/molecule; M, electrophoretic mobility in free solution at 0.1 ionic strength in cm%/V.

AN DER LAN ET AL.

212

TT, freshly iodinated TT appears homogeneous in PAGE (pH 3.7 and 11.0) and in IFPA except for 15% of the label which does not stack in PAGE and a minor peak in electrofocusing gels (component TT,). The minor contamination revealed by PAGE appears tolerable for the purpose of quantitative biological studies, provided one corrects for it. It consists of about 5% of the label, which is of small molecular weight and not bound to the protein, and about lo%, which is associated with protein aggregates. The final step in the iodination procedure, gel filtration, does not have sufficient resolving capacity to remove these contaminants on columns of practical dimensions. PAGE removes these contaminants effectively, presumably because it employs a more restrictive, closely fitting, and obligatory molecular sieve (30), and because the electric field may actively remove charged adsorbed material from the macromolecule. When iodinated TT, which has been stored after iodination in either liquid nitrogen or at -14”C, is analyzed by IFPA, a second protein component, designated as TT2, is found. PAGE, under the conditions used, does not reveal TT,. The heterogeneity of 1251-labeled TT in IFPA, in contrast to that in PAGE, is major; after a storage time of several weeks TT2, which has a pl’ of about 5.8, replaces at least one-half of the protein with a PI’ or 5.1. Neither PAGE nor IFPA can account in physical and molecular terms for the significant differences in bioactivity of TT iodinated by the Bolton-Hunter and the HunterGreenwood methods. TABLE PHYSICAL ANIONIC

CHARACTERISTICS OF STANDARD PROTEINS

Protein (molecular weight) BSA monomer (67,000) BSA dimer (134,000) Aldolase (161,000) Phosphorylase b (185,000) Ferritin (450,000) Thyroglobulin (669,000)

-.

IIIA

YO

MO x 10-S

v

3.76 3.77 1.53 2.79 4.87

9.9 9.5 3.8 6.9 1.2

17.6 24.8 11.0 21.6 59.9

8.98

2.1

136.5

TABLE PHYSICAL

CHARACTERISTICS OF CATIONIC STANDARD PROTEINS

Protein (molecular weight) Ribonuclease a (12,700) Myoglobin (17,800) Chymotrypsin (25,100) Hemoglobin (64,450) y-Globulin (160,000)

Pur$cation

IIIB

Yll

M,

x 10-S

1.42 1.22 1.41

6.5 5.5

1.77

7.2 8.2

2.11

6.1

v

4.7 4.8 6.4 12.6 23.5

of 1251-Labeled TT

For use in biological experiments requiring a homogeneous protein, 1251-labeled TT can be separated by PAGE from 15% iodinated contaminants. The toxin can be eluted from the resolving gel by diffusion. This procedure is sufficient for purifying toxin immediately after iodination. However, it does not have the resolving capacity (at an extreme of pH to which one is restricted, due to the low mobility of 1251-labeled TT) to remove the contamination by component TT, which increases with time of storage. To prepare homogeneous TT1, TT2 needs to be removed from stored 1251-labeled TT by IFPA. Data obtained with iodinated toxin containing TT2, as well as TT1, may be difficult to interpret. Put before the alternative of iodinating tetanus toxin de novo just prior to use or of purifying stored iodinated toxin by IFPA immediately prior to each experiment, the latter approach may be preferable. However, quantitative recovery of TT from IFPA slices remains a problem. Using diffusion 50% recovery of label was possible. However, only at high loads was bioactivity retained. Electrophoretic elution of low loads was attempted but, in contrast to other proteins (27), TT seems be exceptionally insoluble in its isoelectric state. Further studies to obtain a simple recovery procedure for small loads are in progress. Mobility

of 1251-Labeled TT

Unless one titrates 1251-labeled TT to pH 9.6, more than 4 pH units above its isoelectric point, it does not migrate electrophoretically at a rate sufficient to reach lower stacking

Y-TETANUS

TOXIN

IN GEL ELECTROPHORESIS

‘r

L

10

20 Ekctrofocuring

40

30 Time

I 50

ov3"rsl

FIG. 4. Gel electrofocusing patterns and pZ’ values of fresh L251-labeled TT: Electrofocusing pZ range 3.5 to 10. Catholyte 0.1 M arginine, anolyte 0.1 M acetic acid. Temperature 0-4°C 200 V. Protein load 60,000 to 100,000 cpm (30 ng TT) of iodination mixture. The shaded areas refer to the fraction designated as TT, (see section 5 of Results). (a) Gel patterns and recovery values; % recovery refers to the cpm per l-mm gel slice divided by the cpm in the whole gel. (b) pH values at which the peaks of TT, and TT, focus after different focusing time periods. Closed symbols: TT,. Open symbols: TTI.

limits of between 0.025 and 0.093 RM units.4 By comparison, ovalbumin stacks 2.8 pH units above its pI of 4.6 and at a lower stacking limit of 0.086 [Fig. 5 of Ref. (23)]. Low constituent mobility suggests that TT has either a very high molecular weight, or an unusually low surface charge density, or is basic. The Ferguson plots showed that the molecular weight of TT is well below 5 x 105, which would be required to substantially “restrict” the protein at the gel concentration used (22), thus excluding the first possibility. The charge density calculated from PAGE data is substantially lower than expected of a protein with a pZ’ of 5.1 on the 4 The free mobility for native TT at pH 8.6 previously derived from free-moving boundary electrophoresis was 2.8 x 1O-5 (29), and is in good agreement with the data derived from analvtical -* ~~~~ PAGE.~-

213

basis of a comparison with standard proteins. However, the amino acid composition reported for a number of preparations of TT, and upon which most authors essentially agree (lo), does not suggest an unusually low content of acidic functional groups. To explain the exceptionally low surface charge density, one would have to make the rather improbable assumption that the polar groups were mostly buried on the inside of the molecule. Evidence supporting a normal hydrophobicity for TT would contradict such a supposition (31). lz51-Labeled TT is not a basic protein. Although its anionic charge density is less than that of a very basic protein [aldolase, p1 = 9.5 (32)], low negative charge density does not necessarily imply a high density of positively charged groups. Neither the amino acid composition (10) nor the results of electrofocusing, where TT, and TTP exhibit pi’s of 5.1 and 5.9, nor the low charge density of lz51-labeled TT in acid, compared to that of the basic standard proteins, support basicity. Only the mobility of TT in an acid milieu seems to indicate that TT is a basic protein since it is capable of migrating at a substantial rate at pH 4.88, which is only 0.2 pH units from its measured pZ’ (or 1.0 pH unit from that of component TT,). This rapid migration in acid is unexplained. It might be due to cationic ligands or to a conformational change,

FIG. 5. The effect of storage time on the relative amount of component TT,. (a) IFPA patterns after various times of storage at -196°C. Electrofocusing conditions as in Fig. 4. (b) Recoveries of component TT, (shaded areas) and TT1; % recovery defined in Fig. 4. Closed symbols: TT,. Open symbols: TT,.

214

AN

DER

L ANE

by which acidic groups are buried, as was discussed above to explain the relatively low migration rate in alkali. The Nature

of Component

TT,

Because TT, is bioactive, we assume that it is an “isotoxin” of TT. TT, must be derived from TT, because it is formed at the same rate at which TT, disappears. TT, is unlikely to be a product of enzyme degradation because it forms progressively at - 196°C. It is also unlikely that TT, is a subunit of TT, because the molecule would only be expected to cleave under reducing conditions (33), which did not prevail. TT, may be formed as a consequence of reactions with free radicals generated in the solvent by y-irradiation. Alterations in the structure of human growth hormone (34) and in the structure of DNA (35,36) have been observed as a consequence of such free radical reactions. ACKNOWLEDGMENTS We are grateful Robbins for critical

to Rachel Schneerson end John review of the manuscript.

11. 12. 13. 14. 15.

16.

17.

18. 19. 20. 21.

REFERENCES 1. SHERMAN, M. R., TUAZON, F. B., DIAZ, S. C., AND MILLER, L. (1976) Biochemistry 15, 980989. 2. LANG, U., KAHN, C. R., AND CHRAMBACH, A. (1980) Endotinology 166, 40-49. 3. VAN HEYNINGEN, W. E. (1974) Nature (London) 249.415-417. 4. MELLANBY, J., AND POPE, D. (1976) in Ganglioside Function. Biochemical and Pharmacological Implications (Porcellati, G., Ceccsrelli, B., and Tettamenti, G., eds.), pp. 215-229, Plenum, New York/London. 5. DIMPFEL, W., HUANG, R. T. C., AND HABERMANN, E. (1977) .I. Newochem. 29,329-334. 6. LEDLEY, F. D., LEE, G., KOHN, L. D., HABIG, W. H., AND HARDEGREE, M. C. (1977) J. Bill. Chem. 252,4049-4055. 7. LEE, G., CONSIGLIO, E., HABIG, W., DYER, S., HARDEGREE, C., AND KOHN, L. D. (1978) Biothem. Btiphys. Res. Commun. 83, 313-320. 8. HABERMANN, E. (1970) Naunyn-Schmiedeberg’s Arch. Phunnakol. 267, 1-19. 9. HABERMANN, E., WELLH~NER, H. H., AND R~ER, K. 0. (1977) Naunyn-Schmiedeberg’s Arch. Pharmukol. 299, 187-196. 10. BIZZINI, B. (1976) in The Specificity and Action

22.

,T AL. of Animal, Bacterial and Plant Toxins (Cuatrecsses, P., ed.), pp. 175-218, Champan & Hall, London. DIMPFEL, W., AND HABERMANN, E. (1977) J. Neurochem. 211, 1111-1120. ROGOL, A. D., AND CHRAMBACH, A. (1975) Endocrinology 97, 406-417. BOLTON, A. E., AND HUNTER, W. M. (1973) Biochem. J. 133, 529-539. GREENWOOD, F. E., HUNTER, W. M., AND Gu)VER, J. S. (1963) Biochem. J. 89, 114-123. CHRAMBACH, A., JOVIN, T. M., SVENDSEN, P. J., AND RODBARD, D. (1976) in Methods of Protein Separation (Catsimpoolss, N., ed.), Vol. 2, pp. 27-161, Plenum, New York.’ RIGHETTI, P. G., AND DRYSDALE, J. W. (1976) in Isoelectric Focusing (Righetti, P. G., and Drysdale, J. W., eds.), North-Holland/American Elsevier, Amsterdam/New York. CHRAMBACH, A., AND SKYLER, J. D. (1974) in XXII Annual Colloquium on Protides of the Biological Fluids (Peeters, H., ed.), pp. 701-713, Pergamon, Oxford. * NEWBY, A. C., MATTHEWS, G., AND CHF~AMBACH, A. (1978) Anal. Biochem. 91, 473-480. JOVIN, T. M. (1973) Biockemistq 12,871-898. JOVIN, T. M. (1973) Ann. N. Y. Acud. Sci. 209, 477-496. JOVIN, T. M., DANTE, M. L., AND CHRAMBACH, A. (1970) Muitiphasic Buffer Systems Output, National Technical Information Service, Springfield, Va. 22151, PB Numbers 196085 to 196091, 259309 to 259312, 203016. BAUMANN, G., AND CHRAMBACH, A. (1976) Anal.

Biockem. 70,32-38. 23. RODBARD,

D., AND CHRAMBACH,

A. (1971)

Ad.

Biochem. 40, 95-131. 24. RODBARD, D., AND CHRAMBACH, A. (1974) in Electrophoresis and Isoelectric Focusing on Polyacrylamide Gel (Allen, R. C., and Maurer, H. R., eds.) pp. 28-61, de Gruyter, Berlin.’ D. (1976) in Methods of Protein 25. RODBARD, Separation (Catsimpoolss, N., ed.), Vol. 2, pp. 145-218, Plenum, New York. B. E., NGUYEN, N. Y., AND 26. CHIDAKEL, CHRAMBACH, A. (1977) Anal. Biochem. 77, 216-225. 27. NGUYEN, N. Y., AND CHRAMBACH, A. (1979) J. B&kern. Biophys. Methods 1, 171-187. N. Y., MCCORMICK, A. G., AND CHRAM28. NGUYEN, BACH, A. (1978) Anal. Biochem. 88, 186-195. 29. PILLEMER, L., WITPLER, R. G., BURRELL, J. I., AND GROSSBERG, D. B. (1948) J. Exp. Med. 88, 205-221. 30. RODBARD, D. (1976) in Methods of Protein Sepsration (Catsimpoolss, N. ed.), Vol. 2, pp. 145- 179, Plenum, New York.

‘=I-TETANUS

TOXIN

IN GEL

31. ROBINSON, J. P., PICKLESIMER, J. E., AND PUETT, D. (1975) J. Biol. Chem. 250, 7435-7442. 32. RIGHETTI, P. G., AND CARAVAGGIO, T. (1976) J. Chromatogr. 127, l-28. 33. MATSUDA, M., AND YONEDA, M. (1974) Biochem. Biophys. Res. Commun. 57, 1257-1262. 34. HOUGHTEN, R., AND CHRAMBACH, A. (1979)Arch. Biochem. Biophys. 197, X3-169. 35. ROGAN, E., ROTH, R., KATOMSKI, P., BENDERSON,

ELECTROPHORESIS

J, AND CAVALIERI, E. (1978) Chem. Interact. 22, 35-51. 36. ROGAN, E. G., KATOMSKI, P. A., ROTH, AND CAVALIERI, E. L. (1979) J. Biol. 205, 755-759. 37. KAPADIA, G., AND CHRAMBACH, A. (1972) Biochem. 48, 90-102. 38. AN DER LAN, B., AND CHRAMBACH, A. Electrophwesis 1, in press.’

215 Biol. R. W., Chem. Anal. (1980)