164, 571-574 (1974)
W. CRAIG CLARKE,* TETSUO HAYASHIDA, Hormone
CHOH HA0 LI
Received April 23, 1974 Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH,-terminal fragment (residues l-134). In the microcomplement-fixation and radioimmunoassay experiments, the NH,-terminal plasmin fragment (residues 1-134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141-191). This, in turn, was more active than the synthetic fragment (residues 95-136).
Digestion of human growth hormone (HGH)” with plasmin has been reported to increase its potency in the pigeon crop-sac assay (1-4) but does not alter its metabolic actions in the rat and man (5). Two biologically active fragments obtained from human plasmin digests of HGH have recently been isolated and partially characterized (6). The synthesis of a biologically active peptide corresponding to the amino acid residues 95-136 in the HGH molecule (7) has also been described (8). This paper reports the immunochemical properties of these three peptide fragments of HGH. EXPERIMENTAL Growth hormone was isolated from human pituitary glands by the method of Li et al. (9). Reducedcarbamidomethylated HGH was prepared by the method of Bewley and Li (10). Procedures ror the preparation of the major product of plasmin digestion of HGH (PL-HGH) and the two active fragments have been previously described (6). PL-HGH was composed of the NH,-terminal residues 1-134 and the COOH-terminal residues 141-191 connected by a ’ Paper 41 in the Human Growth Hormone series. 2Recipient of a postdoctoral fellowship from the Medical Research Council of Canada. 3Abbreviations used: HGH, human growth hormone; RA-HGH, reduced-carbamidomethylated human growth hormone; PL-HGH, plasma-treated human growth hormone.
disulfide linkage between positions 53 and 165. Reduction and carbamidomethylation of PL-HGH yielded the two fragments: RA-HGH-(l-134) and RA-HGH-(141-191). Nn-Acetyl-HGH-(95-136) was synthesized as previously described (8). Antiserum to HGH was prepared by the immunization of guinea pigs with a total of 1 mg antigen over a period of 6 wk as previously described for porcine prolactin (11). The gel double-diffusion technique of Ouchterlony (12) was performed using 1% agar in 0.01 M phosphate buffer (pH 7.5). Microcomplement fixation was performed according to Wasserman and Levine (13). For radioimmunoassay, the double-antibody procedure of Schalch and Reichlin (14) was employed with slight modifications. Highly purified HGH (9) was used for the preparation of standards and labeling’ with 13’1 according to the method of Greenwood et al. (15). The labeled hormone was further purified on the day of assay by chromatography on Sephadex G-100. The separation of antibody-bound from free [‘3LI]HGH was accomplished by immunoprecipitation with rabbit antiserum to guinea pig gamma globulin. RESULTS
Antisera to HGH produced a single, sharp precipitin line against 0.5 pg of HGH. PL-HGH, RA-HGH, and RAHGH-(1-134) also gave a single precipitin line which showed a reaction of qualitative identity with the native hormone. RAHGH-(141-191) and N”-acetyl-HGH-(95 4Iodinations were performed through the courtesy of Drs. S. Kaplan and M. M. Grumbach.
136) failed to give a precipitin reaction. Results of some of the immunodiffusion studies are shown in Fig. 1. Antiserum GP 12 at a dilution of 1:2000 reacted with 100 ng HGH to fix about 85% complement (Fig. 2A). At the same dilution of antiserum, RA-HGH reacted slightly less than did the native hormone, whereas PL-HGH fixed about one-third less complement at 100 ng (Fig. 2A). In another assay with antiserum GP 12 (Fig. 2B) HGH fixed 66% complement at 100 ng, RA-HGH-( 1-134) fixed 60% complement at 500 ng, and RA-HGH-(141-191) fixed increasing amounts of complement up to 2 pg antigen. W-Acetyl-HGH-(95-136) reacted with antiserum GP 12 to fix 40% complement at 100 ng compared to fixation of 83% complement by the same amount of HGH (Fig. 2C). Antiserum GP I diluted l:lO,OOOreacted with 20 ng HGH to fix 75% complement while RA-HGH-(1-134) fixed 35% complement at 500 ng, RA-HGH-(141-191) fixed 57% complement at 2 pg, and W-acetyl-
HGH-(95-136) fixed only 16% complement at 1 pg. Similarly, using antiserum GP 29 diluted l:lO,OOO, HGH reached equivalence at 50 ng to fix 98% complement, RA-HGH-( 1-134) reached equivalence at 100 ng to fix 34% complement, W-acetylHGH-(95-136) reached equivalence at 300 ng to fix 17% complement but RAHGH-( 141-191) showed no definite equivalence point up to 500 ng antigen. Antiserum GP 15 gave similar results with the plasmin fragments, but like GP 12 it reacted more strongly with the synthetic fragment that did GP 1. In the radioimmunoassay studies, PLHGH and RA-HGH gave inhibition curves with antiserum GP 28 which were parallel to and quite close to that of HGH (Fig. 3). These results were in general accord with those obtained by complement-fixation experiments. It can be seen from Fig. 4 that RA-HGH-( l-134) showed a relatively strong inhibition curve that was likewise parallel to that of HGH. Although RAHGH-( 141-191) also gave a parallel inhibition curve, it was much less effective than the NH,-terminal fragment in this regard, requiring considerably higher antigen concentrations to give the same degree of inhibition. The synthetic fragment, Wacetyl-HGH-(95-136), showed a slight, but significant, ability to compete in this system, as indicated by the relatively flat inhibition curve. Several other assays performed with antisera GP 15 and GP 29 yielded results very similar to those described here. DISCUSSION
FIG. 1. Immunodiffusion plate showing precipitin reactions of HGH, PL-HGH, RA-HGH, the NH,-terminal plasmin fragment (l-134), and the COOH-terminal fragment (141-191) with antiserum to HGH (50 pl antiserum GP 28 in the center well). Note absence of any visible precipitin line with the COOH-terminal plasmin fragment. Photographed after 24-hr diffusion.
Both the microcomplement-fixation and radioimmunoassay data presented here suggest that RA-HGH-(1-134) retains a significant number of the immunoreactive determinants of HGH. The possibility that our HGH fragments were contaminated with undigested protein which could be responsible for the observed immunoreactivity was considered unlikely. RAHGH-(1-134) had much greater immunoreactivity than could be accounted for on the basis of contamination with
HGH. In the agar diffusion studies, even
OF HGH FRAGMENTS
100 500 2000
FIG. 2. A. Microcomplement-fixation curves obtained with HGH (O-e), RA-HGH (Cl-•), or PL-HGH (A-A) and antiserum GP 12 diluted l/2000. B. Microcomplement-fixation curves obtained with HGH (a-O), RA-HGH-(1-134) (A-A), or RA-HGH-(141-191) (W--W) and antiserum GP 12 diluted l/2000. C. Microcomplement-fixation curves obtained with HGH (0-O) or N”-acetyl-HGH-(95-136) (O-O) and antiserum GP 12 diluted l/2000.
FIG. 3. Competition of HGH ([email protected]
), RAHGH (A-A), and PL-HGH (A-A) in the HGH radioimmunoassay system. Final dilution of antiserum GP 28 was l/1,600,000.
100 pg RA-HGH-(141-191) failed to produce a precipitin reaction, whereas only 0.5 pg of HGH was sufficient to give a detectable precipitin line. When compared to HGH, RA-HGH retains virtually full biological activity (16, 17) and most of its immunoreactivity as herein described. Earlier radioimmunoassay studies (18) have already shown that reduction and carbamidomethylation of HGH does not abolish its immunological activity.
FIG. 4. Competition of HGH (O-O), RAHGH - (1-134) (A-A), RA - HGH - (141-191) (A-A), and N”-acetyl-HGH-([email protected]
(L-0) in the HGH radioimmunoassay system. Final dilution of antiserum GP 28 was l/1,500,000.
Plasmin digestion of HGH (PL-HGH) caused a 30% decrease in the maximal amount of complement fixed, but did not shift the equivalence point laterally (Fig. 2A). Similar changes have also been observed after boiling HGH in 8 M urea at pH 7.5 (19). The “vertical shift” of the equivalence point has been explained by diminished reactivity of a few antigenic determinants (20). On the other hand, the “lateral shift” of the equivalence point for RAHGH-(1-134) to 500 ng indicates a greater
decrease in affinity for the antiserum. RAHGH-(141-191) failed to exhibit a definite equivalence point but continued to fix complement up to very high antigen concentrations. A similar pattern was obtained with HGH linked to Sepharose gel
AND LI ACKNOWLEDGMENTS
We thank Eleanor Lasky and Daniel Gordon for technical assistance. The work was supported in part by the National Institutes of Health (HD 04963) and the American Cancer Society.
It is of interest that in the radioimmunoassay studies (Fig. 4) both the RAHGH-( 1-134) and RA-HGH-( 141-191) fragments gave inhibition curves that were parallel to and, therefore, presumably qualitatively identical to HGH on the basis of their immunoreactivities. The parallel inhibition curves given by the plasmin fragments in the radioimmunoassay are not due to HGH contamination because both fragments yielded complement-fixation curves (Fig. 2B) with distinctive shapes. Thus, our comparative study has shown that antigens giving parallel inhibition curves in radioimmunoassay are not necessarily identical. It is also evident that microcomplement fixation is more sensitive to antigenic differences than is radioimmunoassay. The lower specificity of radioimmunoassay may result at least in part from alteration of the HGH standard by introduction of the iodine label. Since RA-HGH and PL-HGH largely retained immunological potency (Figs. 2A and 3), we believe that the diminished immunoreactivity of RA-HGH-( 1-134) and RA-HGH-(141-191) (Figs. 2B and 4) is not the result of the chemical modifications per se, but of the loss of native conformation suffered through cleavage of the HGH molecule. In addition to its greater immunoreactivity, the NH,-terminal portion of the molecule has more biological activity than either the COOH-terminal portion (6) or N”-acetyl-HGH (95136) (8). N”-Acetyl-HGH-(95-136) showed considerable complement-fixing activity (Fig. 2C) with antisera GP 12 and GP 15, although it reacted with GP 1 to a lesser extent. However, its rather flat inhibition curve in the radioimmunoassays (Fig. 4) indicated a much lower affinity for the antibody than was observed for the plasmin fragments, and certainly less than that indicated by microcomplement fixation.
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