Heterogeneity of Purified Human Pituitary Luteinizing Hormone: Effect on Hormone Measurement by Radioimmunoassay R. BENVENISTE, L. A. FROHMAN, AND D. RABINOWITZ Department of Chemical Endocrinology, Hadassah University Hospital, Jerusalem, Israel assay, and maximum activity appeared to correspond to molecules with elution characteristics similar to the unlabelled molecule. All fractions of peak II were immunologically active when tested against rabbit anti-hCG in excess. Two immunoassay systems with improved specificity for hLH and hLH-a, respectively, have been employed to show the presence of a subunit in one of the hLH preparations. These data have relevance with respect to testing of immuno and receptor potencies of hLH preparations. (Endocrinology 97: 20, 1975)
ABSTRACT. Purified preparations of human luteinizing hormone (hLH), Hartree IRC-2, and NIH LER-960, have been examined after gel filtration of unlabelled and iodinated hormone. Several peaks of radioactivity were observed corresponding to dimeric (peak I), monomeric (peak II), and sub-unit (peak III) hLH forms. The immunologic and receptor activities of each fraction have been evaluated. Receptor activity was found in peaks I and II but not in peak III. Within peak II all fractions were not equally active in the receptor
T
of antibody, based on successive adsorption of the antiserum by a and /3 subunits of hCG coupled to Sepharose (2). By selecting highly purified tracers of intact hormone or individual a subunit and appropriate dilutions of the mixed antiserum, we have developed two immunoassays which differentiate between hLH and hLH-a with a high degree of selectivity (3). We have used these assays together with a) appropriate techniques for determination of physical constants and b) a radioligand receptor assay using a particulate fraction of rat testis, to characterize two highly purified preparations of human LH.
HE specificity of the radioimmunoassay (RIA) necessitates the use of highly purified hormone preparations as radioactive tracers, since most antisera in use contain more than one population of antibody. The present study arose from our earlier studies (1) which revealed fluctuations of the immunologic potency of one highly purified human luteinizing hormone (hLH) preparation (Hartree, IRC-2) relative to the international reference preparation (2nd IRP-HMG), depending upon a) the dilution of the antiserum employed in the RIA, and b) the aliquot chosen for tracer after Sephadex gel chromatography of the labelled hormone (IRC-2). In order to elucidate this observation, the nature of the antiserum employed and of highly purified hLH preparations were successively investigated. We have recently presented evidence that rabbit anti-human chorionic gonadotropin (hCG) antiserum prepared by Dr. S. W. Rosen contains at least two populations
Materials and Methods Pituitary hLH preparations. Two preparations of purified hLH suitable for immunoassay iodination were used: IRC-2, kindly donated by Dr. A. S. Hartree, and LER-960, a gift of the National Institutes of Health. The stated potency for IRC-2 by the ovarian ascorbic acid depletion assay (OAAD) was approximately 5 x that of NIH-LH SI (4). The activity of 1 mg of NIH-LH SI has been reported to be equivalent (OAAD) to 588 IU, 2nd IRP-HMG (5). In the radio-ligand receptor assay described below, 1 mg of the IRC-2 preparation is equipotent to 2,000 IU, 2nd IRP-HMG (6). The ratio between
Received February 14, 1974. Dr. Benveniste's and Dr. Frohman's present address is: Division of Endocrinology and Metabolism, Department of Medicine, Michael Reese Medical Center, Chicago, Illinois.
20
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RIA AND hLH HETEROGENEITY immuno and receptor activity of IRC-2 was originally reported to be approximately four, when compared with the 2nd IRP-HMG (6). As a result of variations in the RIA dependent upon the extent of purification of. the tracer (IRC-2) and the dilution of the antiserum (Rosen antihCG antiserum) a ratio of about two has been more recently observed. The stated potency for LER-960 was 923 IU, 2nd IRP-HMG/mg (OAAD) but a higher value has been ascribed to this preparation by Rosemberg et al. (1,465 IU/mg) (5) and by Leidenberger and Reichert (2,990-7,740 IU/mg) (7). In our receptor assay, 1 nig of LER-960 was equipotent to 1,700 IU, 2nd IRP-HMG and the ratio between immuno and receptor activity was found to be approximately one (unpublished data). Iodination procedure. The method of Greenwood et al. (8) was employed. Specific activities ranging between 7 and 40 ixCV/xg were obtained. Separation of 125I-bound protein from free iodide was achieved by filtration over Sephadex G-75. Radio-ligand receptor assay. We have reported elsewhere the details of this assay (6). In brief, the particulate fraction of rat testis homogenate sedimenting at 15,000 rpm following differential centrifugation in Tris-sucrose buffer, was incubated for 48 h at 4 C in the presence of labelled hormone. Labelled hormone bound to the receptor was separated from unbound tracer by high speed centrifugation. The receptor activity of the tracer was defined as the percent of counts bound to the receptor (total binding) minus the percent of counts bound when an excess of cold hormone, 5 IU of hCG, was present (nonspecific binding). Gel filtration. Samples were filtered on Sephadex G-100 (2.2-2.5 X 85-90 cm) by the descending technique at a flow rate of 24 ml/h. The eluting buffer was 0.01M phosphate-0.01% merthiolate, pH 7.6, to which 0.1% bovine serum albumin (BSA, Armour, Fraction V), was added for some experiments. Fractions of 4 ml were collected. Elution positions were characterized for each peak by the partition coefficient between the liquid phase and the gel phase (KaV) calculated Ve - Vo from the equation (9): KaV = — in which Ve *t
'0
21
is the peak elution volume of the material studied; Vo is the peak elution volume of Blue Dextran (Pharmacia), and Vt, the total volume of the gel bed obtained by calibration of the chromatographic tube. Stokes radius determination by gel filtration. Values for Stokes radii were obtained as recommended by Siegel and Monty (10), with the modification that Kav, instead of the partition coefficient between the mobile liquid phase and the stationary liquid phase (K w = 3.87) and lysozyme (S2o, w = 1-79) (Nutritional Biochemical Corp). Each run consisted of one tube containing 1 mg of each standard, a second tube containing 0.2 mg of each of the same two standard proteins, and three tubes containing individual labelled samples. [131I]iodoBSA was added to each tube as an internal marker. By this procedure we were able to assess the effect of concentration on sedimentation velocity since we were centrifuging the standard proteins at finite concentration, and the radioiodinated samples at conditions approaching
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BENVENISTE, FROHMAN AND RABINOWITZ
22
"zero" concentration. By plotting the S20, u) of the standards versus the calculated Rf at concentration zero of these standards, where fraction number of standard in each centrifuge tube total number of fractions a linear relation was obtained which was used to calculate the S20, w values of the unknowns after experimental determination of their Rf. IMMUNOASSAY
Molecular weight
Endo • 1975 Vol 97 • No 1
determinations
The molecular weights were calculated using 6rj7r Nas the equation (10): M = in which -n = the 1 —Vp system viscosity, N = Avogadro's number, a = Stokes radius, V = the partial specific volume taken as 0.71 (11), s = the sedimentation value, and p = density of the medium. The limitations of this technique as applied to studies of LH have too
too
90-
to $0-
40 ' 40-
90
as nm
01
os MO
9 10 PC» TUBt
iO
60
MtOO
MO PBt TUBC
M6P€RWBC
FIG. 1. Displacement curves for immunoassays A and B. Data are presented as percentage of bound: free ratio in the presence of unlabelled hormone (B/F)x relative to bound: free ratio in the presence of tracer alone (B/F)o. The horizontal axis shows ng of hormone present per tube. Upper panel: Immunoassay A. Lower panel: Immunoassay B.
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23
RIA AND hLH HETEROGENEITY cpm/Tube MW 120 125 l-hLH(LER96O)
114
21fjCl/fjg
we
FIG. 2. Elution profiles on SephadexG-100(dimensions: 2.2 x 85 cm) of 4 /u,g of LER-960 labelled to a specific activity of 21 /xCi/ fxg. Elution buffer: 0.01M phosphate-0.1% BSA, pH 7.6. Left: Iodination performed with 15 fig of chloramine T. Right: Iodination performed with 30 fig of chloramine T.
1
32
26
24
20
16 • 1
,0 30
0.1, 40
0
0.2 , 0.3 Kgy v 50 3(T TUBE NUMBER
been discussed by Reichert and Jiang (12), Ward and Arnott (13) and Ryan et al. (11).
Radioimmunoassays.
The rabbit anti-hCG
serum employed in this study contains two populations of antibody: Population I which recognizes hCG (and also hLH) and Population II which recognizes determinants of the a subunit of hCG (and hLH-a). The titer of population I is higher than that of Population II. Using purified [125I]iodo-hLH and [125I]iodo-hCGa and an appropriate dilution of the antiserum two immunoassay systems were developed. Immunoassay A. This assay consisted of purified [125I]iodohLH (Kav = 0.24; see Figs. 3 and 4) and Rosen anti-hCG antibody (final concentration: 1.2 x 10"6). At this high dilution, the antibody Population II is effectively diluted out. Figure 1 (upper panel) shows that this system recognizes hCG (Canfield, CR 117), hLH (LER-960 and IRC-2) and p subunit preparations (hCG-/3, Canfield CR 117, and hLH-0, Medical Research Council (MRC), Mill Hill, London, Code No. 71/342) with a high degree of sensitivity. In contrast, the recognition of hCG-a (Canfield CR 117), hFSH-a (LER-1661-8/10) and (not shown) hLH-a (MRC, 72/70) occurs only at 20-200 x greater concentrations.
50
Immunoassay B. This assay employs purified [125I]iodo-hCG-a and Rosen anti-hCG antibody at a higher final concentration and as shown in Fig. 1, lower panel, recognizes hCG-a and hLH-a with a high degree of sensitivity. At this dilution, the excess of Population I antibodies binds hCG and hCG-/3 which do not compete with [125I]iodo-hCG-a for Population II antibodies. The competition shown by the two preparations of hLH (IRC-2 and LER-960) and by hLH-/3 (at 20, 300, and 50 x greater concentrations, respectively) demonstrates parallelism to hCGa and is likely due to the presence of the a subunit. As indicated by the MRC, the hLH-/3 preparation was not tested for hLH-a presence prior to distribution. A 2 - 5 % contamination of the hLH-/3 with hLH-a could explain its behavior in this immunoassay system. Evidence for the presence of the a subunit in the hLH IRC-2 preparation will be presented below.
Results Gel filtration of labelled human luteinizing hormone Samples (4 fig) of hLH (LER-960) were iodinated using either 15 fig (No. 1) or 30 fig (No. 2) of chloramine T. On both
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24
BENVENISTE, FROHMAN AND RABINOWITZ
Endo i 1975 Vol 97 . No 1
peared within the void volume, 23% in peak I (Kav = 0.10), and 56% in peak II 14.000 (Kav = 0.24). Most of the radioactivity from iodination No. 2 appeared in the void 12,000 volume (right side of Fig. 2) and in a region including peak I, while approximately 10% 10.000 of the radioactivity was present in peak II. Recoveries of total radioactivity after gel filtration ranged between 90 and 110%. 6.000 The elution profile of the iodinated IRC-2 preparation (Fig. 3, upper panel) /// 6000 contained a void volume peak as well as three further peaks: Peak I (Kav = 0.10), 4000 peak II (Kav = 0.24) and a broader peak III (Kav = 0.40). Aliquots corresponding to Kav = 0.10, 0.24 and 0.40 from peak I, peak 2000 II and peak III respectively, were refiltered and the positions of the three 0 peaks remained constant (Fig. 3, lower 50 40 cpm panel). Peak III has been consistently 600 found following iodination of IRC-2 over a wide range of specific activities. The immunologic and receptor activity of 600 peaks I, II and III of iodinated IRC-2 are shown in Fig. 4. The lower panel illus400 trates the distribution of radioactivity on Sephadex G-100. Aliquots of each tube containing 5000 CPM were a) incubated in 200 the presence of excess rabbit anti-hCG (10~3 dilution), and b) tested for receptor L .0.1 . 02 .03. 0.4 . Q5K, 0 av 40 50 60 70 binding activity. These results are shown TUBE NUMBER in the upper panel. While approximately 80% of labelled material from peaks I, II, FIG. 3. Upper panel: Gel filtration on Sephadex G-100 (2.5 x 85 cm) of 4 /xg of IRC-2 labelled to a specific and III were immunologically bound, reactivity of 37 fiCi/fig with 25 /xg of chloramine T. ceptor binding activity was present only in Lower panel: Aliquots of tube numbers 47, 59, and 71 peaks I and II. [125I]iodo-hLH specific activfrom the gel filtration presented in the upper panel ity was determined in the two fractions were rechromatographed. The buffer employed is indicated by the arrows by comparing the described in the legend to Fig. 2. displacement curves of the nonradioactive LH reference preparation with that of occasions, sodium metabisulfite was added increasing quantities of each of the in a 2.5 molar excess. All other conditions, [125I]iodo-hLH fractions and found to be in particular the volumes, were identical. A within 10% of one another. specific activity of 21 fJiCi/fig for both iodinations were observed. Aliquots of the Dissociation of labelled hLH LER-960 labelled products, freed of unreacted iodide, were then filtered on Sephadex Peak II obtained after gel filtration of G-100. iodinated LER-960 was treated with 5M Twenty-one percent of the label from urea overnight at room temperature and iodination No. 1 (left side of Fig. 2) ap- was filtered on Sephadex G-100. A peak of cpm
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RIA AND hLH HETEROGENEITY
25
radioactivity corresponding to monomeric hLH (Kav = 0.24) was observed along with a second peak corresponding to dissociated hLH (Kav = 0.40) which was identical with peak III of the untreated labelled IRC-2 preparation. When IRC-2 peak III was mixed with the latter peak of urea-treated iodinated LER-960 and the mixture filtered on Sephadex G-100, a single peak was obtained (KaV = 0.40).
Data on the physical constants of the various preparations are reported in Table 1. These data are consistent with homology (a) among peak II of LER-960, peak II of IRC-2, and the monomeric hLH form; (b) among peak I of LER-960, peak I of IRC-2, and a dimeric hLH form; and (c) between peak III of IRC-2 and a dissociated hLH form obtained by urea treatment of the iodinated peak II of LER-960.
Determination of physical constants of the different peaks of labelled LER-960 and IRC-2
Gel filtration of non-iodinated IRC-2 preparation
Native pituitary hLH IRC-2 was filtered Determination of Stokes radii of on Sephadex G-100 in order to ascertain molecules in LER-960 and IRC-2 peaks I whether there was evidence of peak III, and II, IRC-2 peak III, and of urea-treated unrelated to iodination. By use of imLER-960, peak II, are illustrated in Fig. 5 munoassay A, which recognizes associated (left panel). There is concordance between hLH and hLH-/3, one broad peak of impeaks I and II for both preparations and munoreactive material was found (Fig. 6). between IRC-2, peak III and the dis- When immunoassay B, selective for hLH-a sociated form of LER-960, peak II after was used, we found immunoreactive mateurea treatment. The relation between S2o,to rial only in a position just prior to labelled and Rf of the standard proteins and labelled peak III, which had been added as a fractions is shown in Fig. 5 (right panel). marker (Fig. 7).
100 Percent tnmunoprecipitabte 80 • — •
Receptor Activity ftrcent DtspKxeable « Binding
0L cpm/tub* M W5
30
40
45
50
55
TUBE NUMBER
FIG. 4. Elution profile of hLH, IRC-2 labelled to a specific activity of 7 /i,Ci//o.g. Radioactive peaks are shown in the lower panel. Receptor activity and percent of label which was immunoprecipitable by antibody excess are shown in the upper panel. [mI]iodo-hLH specific activities were determined in the two fractions indicated by the arrows as described in the results.
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BENVENISTE, FROHMAN AND RABINOWITZ
26
Discussion We have examined two highly purified preparations of hLH, IRC-2 (Hartree) and LER-960 (National Pituitary Agency). These hormones were labelled and subsequently chromatographed on Sephadex G-100 and multiple peaks of radioactivity
090
H"3 Xytochrome 0.80
were observed. What we call peak II in both preparations (KaV = 0.24, mol wt = 33,400), corresponds to monomeric [125I]iodo-hLH,
Chymofryps/nogen
in accord with the detailed study of Ryan et at. (11). We have compared elution profiles of monomeric nonradioactive and of labelled 125I-hLH (Fig. 6). The former eluted
070 Ovalbumin
earlier than the latter which is consistent
LER960 IRC-2 A PeakII
0.60
Endo • 1975 Vol 97 . No 1
with the observation of Ryan et al. (11) that hLH (Stokes radius = 30.4A) is more
rapidly eluted than [131I]iodo-hLH (3O.lA).
0.50
15
Albumin LER-960 IRC-2 20 25 30 35 STOKESRADIUSfA*)
•Peak I
FIG. 5A. Plot of Stokes radius (A) vs the experimentally determined K^^ value for reference proteins, labelled LER-960 and IRC-2 (peaks I and II) and dissociated form of urea treated LER-960 peak II, and untreated IRC-2 peak III.
The fractions comprising peak II were homogeneous with respect to their immunoreactivity in the presence of excess anti-hCG serum (10~3) while significant differences were observed in receptor activity (Fig. 4). No differences in specific activity of the [mI]iodo-hLH could be demonstrated (data not shown) to account for the variations in receptor activity. Further studies will be required to distinguish between these fractions. Peak I is possibly a dimer (KaV = 0.10,
>20w 4
3 2
Dissociated PeakIIofLER960 Pea kill IRC-2
0.1
0.2 P
0.3
0A
0.5
_ Number of fraction of the Sample Total Number of fractions
0.6
FIG. 5B. Sedimentation velocity of standard proteins and of labelled unknowns in 5 to 27 percent sucrose gradient at 31,000 rpm for 40 h. Bovine serum albumin (BSA) and lysozyme (Lys) were centrifuged at two concentrations 50 mg/ml and 10 mg/ml. Values of the R, of these standards at "zero" concentration were calculated and plotted in relation to their S2o,u> obtained from the literature. S20,u> of IRC-2 peaks I, II, III and LER-960 peak I, II and urea treated peak II were determined, assuming the concentration to be zero.
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RIA AND hLH HETEROGENEITY TABLE 1. Physical labelled peaks I, preparation, and of II of hLH LER-960
and hydrodynamic properties of II, and III from hLH IRC-2 peaks I, II and urea-treated peak preparation
Peak I of LER-960 and IRC 2
Stokes radius (A)
27
Peak II of LER-960 and IRC 2
Peak II of LER-960 Urea Treated and IRC 2 Peak III
0.10
0.24
0.40
37.2
29.8
22.6
IMMUNOASSAY A 0.2
o.i
0.3
nghLH/ml of eluate
NUMBER
Sedimentation constant (S,o w)
4.35
2.78
2.05
Molecular weight
65,900
33,400
18,500
FIG. 6. Elution profile of IRC-2 on Sephadex G-100 (2.2 x 85 cm) obtained when 0.4 ml fractions of eluates were assayed in immunoassay A. The arrows show position of markers 125I-hLH (peak II) and 125 I-hLH-
ABSTRACT. Purified preparations of human luteinizing hormone (hLH), Hartree IRC-2, and NIH LER-960, have been examined after gel filtration of unlabelled and iodinated hormone. Several peaks of radioactivity were observed corresponding to dimeric (peak I), monomeric (peak II), and sub-unit (peak III) hLH forms. The immunologic and receptor activities of each fraction have been evaluated. Receptor activity was found in peaks I and II but not in peak III. Within peak II all fractions were not equally active in the receptor
T
of antibody, based on successive adsorption of the antiserum by a and /3 subunits of hCG coupled to Sepharose (2). By selecting highly purified tracers of intact hormone or individual a subunit and appropriate dilutions of the mixed antiserum, we have developed two immunoassays which differentiate between hLH and hLH-a with a high degree of selectivity (3). We have used these assays together with a) appropriate techniques for determination of physical constants and b) a radioligand receptor assay using a particulate fraction of rat testis, to characterize two highly purified preparations of human LH.
HE specificity of the radioimmunoassay (RIA) necessitates the use of highly purified hormone preparations as radioactive tracers, since most antisera in use contain more than one population of antibody. The present study arose from our earlier studies (1) which revealed fluctuations of the immunologic potency of one highly purified human luteinizing hormone (hLH) preparation (Hartree, IRC-2) relative to the international reference preparation (2nd IRP-HMG), depending upon a) the dilution of the antiserum employed in the RIA, and b) the aliquot chosen for tracer after Sephadex gel chromatography of the labelled hormone (IRC-2). In order to elucidate this observation, the nature of the antiserum employed and of highly purified hLH preparations were successively investigated. We have recently presented evidence that rabbit anti-human chorionic gonadotropin (hCG) antiserum prepared by Dr. S. W. Rosen contains at least two populations
Materials and Methods Pituitary hLH preparations. Two preparations of purified hLH suitable for immunoassay iodination were used: IRC-2, kindly donated by Dr. A. S. Hartree, and LER-960, a gift of the National Institutes of Health. The stated potency for IRC-2 by the ovarian ascorbic acid depletion assay (OAAD) was approximately 5 x that of NIH-LH SI (4). The activity of 1 mg of NIH-LH SI has been reported to be equivalent (OAAD) to 588 IU, 2nd IRP-HMG (5). In the radio-ligand receptor assay described below, 1 mg of the IRC-2 preparation is equipotent to 2,000 IU, 2nd IRP-HMG (6). The ratio between
Received February 14, 1974. Dr. Benveniste's and Dr. Frohman's present address is: Division of Endocrinology and Metabolism, Department of Medicine, Michael Reese Medical Center, Chicago, Illinois.
20
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RIA AND hLH HETEROGENEITY immuno and receptor activity of IRC-2 was originally reported to be approximately four, when compared with the 2nd IRP-HMG (6). As a result of variations in the RIA dependent upon the extent of purification of. the tracer (IRC-2) and the dilution of the antiserum (Rosen antihCG antiserum) a ratio of about two has been more recently observed. The stated potency for LER-960 was 923 IU, 2nd IRP-HMG/mg (OAAD) but a higher value has been ascribed to this preparation by Rosemberg et al. (1,465 IU/mg) (5) and by Leidenberger and Reichert (2,990-7,740 IU/mg) (7). In our receptor assay, 1 nig of LER-960 was equipotent to 1,700 IU, 2nd IRP-HMG and the ratio between immuno and receptor activity was found to be approximately one (unpublished data). Iodination procedure. The method of Greenwood et al. (8) was employed. Specific activities ranging between 7 and 40 ixCV/xg were obtained. Separation of 125I-bound protein from free iodide was achieved by filtration over Sephadex G-75. Radio-ligand receptor assay. We have reported elsewhere the details of this assay (6). In brief, the particulate fraction of rat testis homogenate sedimenting at 15,000 rpm following differential centrifugation in Tris-sucrose buffer, was incubated for 48 h at 4 C in the presence of labelled hormone. Labelled hormone bound to the receptor was separated from unbound tracer by high speed centrifugation. The receptor activity of the tracer was defined as the percent of counts bound to the receptor (total binding) minus the percent of counts bound when an excess of cold hormone, 5 IU of hCG, was present (nonspecific binding). Gel filtration. Samples were filtered on Sephadex G-100 (2.2-2.5 X 85-90 cm) by the descending technique at a flow rate of 24 ml/h. The eluting buffer was 0.01M phosphate-0.01% merthiolate, pH 7.6, to which 0.1% bovine serum albumin (BSA, Armour, Fraction V), was added for some experiments. Fractions of 4 ml were collected. Elution positions were characterized for each peak by the partition coefficient between the liquid phase and the gel phase (KaV) calculated Ve - Vo from the equation (9): KaV = — in which Ve *t
'0
21
is the peak elution volume of the material studied; Vo is the peak elution volume of Blue Dextran (Pharmacia), and Vt, the total volume of the gel bed obtained by calibration of the chromatographic tube. Stokes radius determination by gel filtration. Values for Stokes radii were obtained as recommended by Siegel and Monty (10), with the modification that Kav, instead of the partition coefficient between the mobile liquid phase and the stationary liquid phase (K w = 3.87) and lysozyme (S2o, w = 1-79) (Nutritional Biochemical Corp). Each run consisted of one tube containing 1 mg of each standard, a second tube containing 0.2 mg of each of the same two standard proteins, and three tubes containing individual labelled samples. [131I]iodoBSA was added to each tube as an internal marker. By this procedure we were able to assess the effect of concentration on sedimentation velocity since we were centrifuging the standard proteins at finite concentration, and the radioiodinated samples at conditions approaching
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BENVENISTE, FROHMAN AND RABINOWITZ
22
"zero" concentration. By plotting the S20, u) of the standards versus the calculated Rf at concentration zero of these standards, where fraction number of standard in each centrifuge tube total number of fractions a linear relation was obtained which was used to calculate the S20, w values of the unknowns after experimental determination of their Rf. IMMUNOASSAY
Molecular weight
Endo • 1975 Vol 97 • No 1
determinations
The molecular weights were calculated using 6rj7r Nas the equation (10): M = in which -n = the 1 —Vp system viscosity, N = Avogadro's number, a = Stokes radius, V = the partial specific volume taken as 0.71 (11), s = the sedimentation value, and p = density of the medium. The limitations of this technique as applied to studies of LH have too
too
90-
to $0-
40 ' 40-
90
as nm
01
os MO
9 10 PC» TUBt
iO
60
MtOO
MO PBt TUBC
M6P€RWBC
FIG. 1. Displacement curves for immunoassays A and B. Data are presented as percentage of bound: free ratio in the presence of unlabelled hormone (B/F)x relative to bound: free ratio in the presence of tracer alone (B/F)o. The horizontal axis shows ng of hormone present per tube. Upper panel: Immunoassay A. Lower panel: Immunoassay B.
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23
RIA AND hLH HETEROGENEITY cpm/Tube MW 120 125 l-hLH(LER96O)
114
21fjCl/fjg
we
FIG. 2. Elution profiles on SephadexG-100(dimensions: 2.2 x 85 cm) of 4 /u,g of LER-960 labelled to a specific activity of 21 /xCi/ fxg. Elution buffer: 0.01M phosphate-0.1% BSA, pH 7.6. Left: Iodination performed with 15 fig of chloramine T. Right: Iodination performed with 30 fig of chloramine T.
1
32
26
24
20
16 • 1
,0 30
0.1, 40
0
0.2 , 0.3 Kgy v 50 3(T TUBE NUMBER
been discussed by Reichert and Jiang (12), Ward and Arnott (13) and Ryan et al. (11).
Radioimmunoassays.
The rabbit anti-hCG
serum employed in this study contains two populations of antibody: Population I which recognizes hCG (and also hLH) and Population II which recognizes determinants of the a subunit of hCG (and hLH-a). The titer of population I is higher than that of Population II. Using purified [125I]iodo-hLH and [125I]iodo-hCGa and an appropriate dilution of the antiserum two immunoassay systems were developed. Immunoassay A. This assay consisted of purified [125I]iodohLH (Kav = 0.24; see Figs. 3 and 4) and Rosen anti-hCG antibody (final concentration: 1.2 x 10"6). At this high dilution, the antibody Population II is effectively diluted out. Figure 1 (upper panel) shows that this system recognizes hCG (Canfield, CR 117), hLH (LER-960 and IRC-2) and p subunit preparations (hCG-/3, Canfield CR 117, and hLH-0, Medical Research Council (MRC), Mill Hill, London, Code No. 71/342) with a high degree of sensitivity. In contrast, the recognition of hCG-a (Canfield CR 117), hFSH-a (LER-1661-8/10) and (not shown) hLH-a (MRC, 72/70) occurs only at 20-200 x greater concentrations.
50
Immunoassay B. This assay employs purified [125I]iodo-hCG-a and Rosen anti-hCG antibody at a higher final concentration and as shown in Fig. 1, lower panel, recognizes hCG-a and hLH-a with a high degree of sensitivity. At this dilution, the excess of Population I antibodies binds hCG and hCG-/3 which do not compete with [125I]iodo-hCG-a for Population II antibodies. The competition shown by the two preparations of hLH (IRC-2 and LER-960) and by hLH-/3 (at 20, 300, and 50 x greater concentrations, respectively) demonstrates parallelism to hCGa and is likely due to the presence of the a subunit. As indicated by the MRC, the hLH-/3 preparation was not tested for hLH-a presence prior to distribution. A 2 - 5 % contamination of the hLH-/3 with hLH-a could explain its behavior in this immunoassay system. Evidence for the presence of the a subunit in the hLH IRC-2 preparation will be presented below.
Results Gel filtration of labelled human luteinizing hormone Samples (4 fig) of hLH (LER-960) were iodinated using either 15 fig (No. 1) or 30 fig (No. 2) of chloramine T. On both
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24
BENVENISTE, FROHMAN AND RABINOWITZ
Endo i 1975 Vol 97 . No 1
peared within the void volume, 23% in peak I (Kav = 0.10), and 56% in peak II 14.000 (Kav = 0.24). Most of the radioactivity from iodination No. 2 appeared in the void 12,000 volume (right side of Fig. 2) and in a region including peak I, while approximately 10% 10.000 of the radioactivity was present in peak II. Recoveries of total radioactivity after gel filtration ranged between 90 and 110%. 6.000 The elution profile of the iodinated IRC-2 preparation (Fig. 3, upper panel) /// 6000 contained a void volume peak as well as three further peaks: Peak I (Kav = 0.10), 4000 peak II (Kav = 0.24) and a broader peak III (Kav = 0.40). Aliquots corresponding to Kav = 0.10, 0.24 and 0.40 from peak I, peak 2000 II and peak III respectively, were refiltered and the positions of the three 0 peaks remained constant (Fig. 3, lower 50 40 cpm panel). Peak III has been consistently 600 found following iodination of IRC-2 over a wide range of specific activities. The immunologic and receptor activity of 600 peaks I, II and III of iodinated IRC-2 are shown in Fig. 4. The lower panel illus400 trates the distribution of radioactivity on Sephadex G-100. Aliquots of each tube containing 5000 CPM were a) incubated in 200 the presence of excess rabbit anti-hCG (10~3 dilution), and b) tested for receptor L .0.1 . 02 .03. 0.4 . Q5K, 0 av 40 50 60 70 binding activity. These results are shown TUBE NUMBER in the upper panel. While approximately 80% of labelled material from peaks I, II, FIG. 3. Upper panel: Gel filtration on Sephadex G-100 (2.5 x 85 cm) of 4 /xg of IRC-2 labelled to a specific and III were immunologically bound, reactivity of 37 fiCi/fig with 25 /xg of chloramine T. ceptor binding activity was present only in Lower panel: Aliquots of tube numbers 47, 59, and 71 peaks I and II. [125I]iodo-hLH specific activfrom the gel filtration presented in the upper panel ity was determined in the two fractions were rechromatographed. The buffer employed is indicated by the arrows by comparing the described in the legend to Fig. 2. displacement curves of the nonradioactive LH reference preparation with that of occasions, sodium metabisulfite was added increasing quantities of each of the in a 2.5 molar excess. All other conditions, [125I]iodo-hLH fractions and found to be in particular the volumes, were identical. A within 10% of one another. specific activity of 21 fJiCi/fig for both iodinations were observed. Aliquots of the Dissociation of labelled hLH LER-960 labelled products, freed of unreacted iodide, were then filtered on Sephadex Peak II obtained after gel filtration of G-100. iodinated LER-960 was treated with 5M Twenty-one percent of the label from urea overnight at room temperature and iodination No. 1 (left side of Fig. 2) ap- was filtered on Sephadex G-100. A peak of cpm
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RIA AND hLH HETEROGENEITY
25
radioactivity corresponding to monomeric hLH (Kav = 0.24) was observed along with a second peak corresponding to dissociated hLH (Kav = 0.40) which was identical with peak III of the untreated labelled IRC-2 preparation. When IRC-2 peak III was mixed with the latter peak of urea-treated iodinated LER-960 and the mixture filtered on Sephadex G-100, a single peak was obtained (KaV = 0.40).
Data on the physical constants of the various preparations are reported in Table 1. These data are consistent with homology (a) among peak II of LER-960, peak II of IRC-2, and the monomeric hLH form; (b) among peak I of LER-960, peak I of IRC-2, and a dimeric hLH form; and (c) between peak III of IRC-2 and a dissociated hLH form obtained by urea treatment of the iodinated peak II of LER-960.
Determination of physical constants of the different peaks of labelled LER-960 and IRC-2
Gel filtration of non-iodinated IRC-2 preparation
Native pituitary hLH IRC-2 was filtered Determination of Stokes radii of on Sephadex G-100 in order to ascertain molecules in LER-960 and IRC-2 peaks I whether there was evidence of peak III, and II, IRC-2 peak III, and of urea-treated unrelated to iodination. By use of imLER-960, peak II, are illustrated in Fig. 5 munoassay A, which recognizes associated (left panel). There is concordance between hLH and hLH-/3, one broad peak of impeaks I and II for both preparations and munoreactive material was found (Fig. 6). between IRC-2, peak III and the dis- When immunoassay B, selective for hLH-a sociated form of LER-960, peak II after was used, we found immunoreactive mateurea treatment. The relation between S2o,to rial only in a position just prior to labelled and Rf of the standard proteins and labelled peak III, which had been added as a fractions is shown in Fig. 5 (right panel). marker (Fig. 7).
100 Percent tnmunoprecipitabte 80 • — •
Receptor Activity ftrcent DtspKxeable « Binding
0L cpm/tub* M W5
30
40
45
50
55
TUBE NUMBER
FIG. 4. Elution profile of hLH, IRC-2 labelled to a specific activity of 7 /i,Ci//o.g. Radioactive peaks are shown in the lower panel. Receptor activity and percent of label which was immunoprecipitable by antibody excess are shown in the upper panel. [mI]iodo-hLH specific activities were determined in the two fractions indicated by the arrows as described in the results.
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BENVENISTE, FROHMAN AND RABINOWITZ
26
Discussion We have examined two highly purified preparations of hLH, IRC-2 (Hartree) and LER-960 (National Pituitary Agency). These hormones were labelled and subsequently chromatographed on Sephadex G-100 and multiple peaks of radioactivity
090
H"3 Xytochrome 0.80
were observed. What we call peak II in both preparations (KaV = 0.24, mol wt = 33,400), corresponds to monomeric [125I]iodo-hLH,
Chymofryps/nogen
in accord with the detailed study of Ryan et at. (11). We have compared elution profiles of monomeric nonradioactive and of labelled 125I-hLH (Fig. 6). The former eluted
070 Ovalbumin
earlier than the latter which is consistent
LER960 IRC-2 A PeakII
0.60
Endo • 1975 Vol 97 . No 1
with the observation of Ryan et al. (11) that hLH (Stokes radius = 30.4A) is more
rapidly eluted than [131I]iodo-hLH (3O.lA).
0.50
15
Albumin LER-960 IRC-2 20 25 30 35 STOKESRADIUSfA*)
•Peak I
FIG. 5A. Plot of Stokes radius (A) vs the experimentally determined K^^ value for reference proteins, labelled LER-960 and IRC-2 (peaks I and II) and dissociated form of urea treated LER-960 peak II, and untreated IRC-2 peak III.
The fractions comprising peak II were homogeneous with respect to their immunoreactivity in the presence of excess anti-hCG serum (10~3) while significant differences were observed in receptor activity (Fig. 4). No differences in specific activity of the [mI]iodo-hLH could be demonstrated (data not shown) to account for the variations in receptor activity. Further studies will be required to distinguish between these fractions. Peak I is possibly a dimer (KaV = 0.10,
>20w 4
3 2
Dissociated PeakIIofLER960 Pea kill IRC-2
0.1
0.2 P
0.3
0A
0.5
_ Number of fraction of the Sample Total Number of fractions
0.6
FIG. 5B. Sedimentation velocity of standard proteins and of labelled unknowns in 5 to 27 percent sucrose gradient at 31,000 rpm for 40 h. Bovine serum albumin (BSA) and lysozyme (Lys) were centrifuged at two concentrations 50 mg/ml and 10 mg/ml. Values of the R, of these standards at "zero" concentration were calculated and plotted in relation to their S2o,u> obtained from the literature. S20,u> of IRC-2 peaks I, II, III and LER-960 peak I, II and urea treated peak II were determined, assuming the concentration to be zero.
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RIA AND hLH HETEROGENEITY TABLE 1. Physical labelled peaks I, preparation, and of II of hLH LER-960
and hydrodynamic properties of II, and III from hLH IRC-2 peaks I, II and urea-treated peak preparation
Peak I of LER-960 and IRC 2
Stokes radius (A)
27
Peak II of LER-960 and IRC 2
Peak II of LER-960 Urea Treated and IRC 2 Peak III
0.10
0.24
0.40
37.2
29.8
22.6
IMMUNOASSAY A 0.2
o.i
0.3
nghLH/ml of eluate
NUMBER
Sedimentation constant (S,o w)
4.35
2.78
2.05
Molecular weight
65,900
33,400
18,500
FIG. 6. Elution profile of IRC-2 on Sephadex G-100 (2.2 x 85 cm) obtained when 0.4 ml fractions of eluates were assayed in immunoassay A. The arrows show position of markers 125I-hLH (peak II) and 125 I-hLH-
