The Reaction


of Biochemistry


of Tetranitromethane Gonadotropin1

ROBERT Department

175, 209-220



and Division of Cell and Molecular Buffalo, Buffalo, New York Received


with Human


OM P. BAHL2 Biology, 14214



of New



7, 1975

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, a and /3, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at a Tyr-88 and/or a Tyr89. Exhaustive nitration of the hormone modified a tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated a subunit obtained by dissociation of the nitrated hormone recombined with the native 6 subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of a Tyr-88 and/or a Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the B subunit, however. Tyr-65 of the a subunit is probably not involved in binding to either the 6 subunit or the hormone receptor. The 6 subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native a to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence. Nitration of the isolated (Y subunit fully converted the remaining tyrosine (Tyr-37) to 3nitrotyrosine in a two-step reaction. The fully nitrated a subunit did not recombine well with the native p subunit and the recombinant hormone has 10% or less of the native activity. Involvement of a Tyr-37 in binding to the 6 subunit is suggested by these data. However, exposure of this residue by a conformational change in the a subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free 6 subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated p subunit did not recombine well with native a but the isolated recombinant had two-thirds of the native activity. From these data we conclude that B Tyr-37 and/or p Tyr-59 are possibly involved in binding to the a subunit but do not have a role in the biological activity. Tyr-82 of p is apparently not involved in either subunit interactions or hormone-receptor binding.

Human chorionic gonadotropin consists of two nonidentical subunits, (Y and /3 (1). 1 This research was supported by grants from United States Public Health Service (HD 08766) the Population Council of New York (M72-39). of us (R. B. C.) is a recipient of a fellowship from Population Council. * To whom all correspondence should be dressed. Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

Two other glycoprotein hormones, thyrotropin and luteinizing hormone, are similarly composed of an (Yand a p subunit (2, 3). The (Y subunits are biologically interchangeable among these hormones while the p subunits are hormone-specific (4). Considerable sequence homology exists among the different p subunits (5). Two of the three tyrosine-containing

the and One the ad209



peptides in hCG-P3 are common to LH-P and TSH-/I. This suggests that these tyrosines might be involved in interactions with the CI type of subunit. A number of studies have been reported on chemical modifications of the glycoprotein hormones (6-11). Modification of tyrosines has been shown to decrease the activities of hCG (6, 7), luteinizing hormone (10, ll), and thyrotropin (11). These considerations of the possible role of tyrosines in the subunit interactions and biological activities of the glycoprotein hormones led us to investigate the effects of nitration on hCG. This report presents data on the reaction of native hCG as well as of its subunits with tetranitromethane. The effects of nitration on subunit interactions and biological and immunological activity are discussed. EXPERIMENTAL


Human chorionic gonadotropin and its subunits : were prepared as previously described (1, 12). Tetranitromethane was from Aldrich Chemical Corp. Trypsin (treated with L-1-tosylamido-2-phenylethyl ketone) was obtained from Worthington. Nitration, isolation of the monomer products, and separation of the subunits. Nitration was carried out in a manner similar to that described by Sokolovsky et al. (13). The protein concentration was 10 PM in 0.05 M Tris-HCl, pH 8.2. Tetranitromethane was added in 95% ethanol as a solution of appropriate concentration to give a final TNM concentration of 0.15 or 2.0 mM when 0.4 vol of TNM solution was added to 100 vol of protein solution. The reaction was carried out at 23°C and the rate was monitored by measuring the absorbance of nitroform anion at 350 nm (c = 14,400 (14)). The reaction was terminated by gel filtration through Sephadex G-25 (coarse) at room temperature with 0.5% NH,HCO, as the eluent. The nitrated protein was lyophilized. Nitrated monomeric form of the hormone was separated from the polymeric products by gel filtration through Sephadex G-100 at 4°C with 0.5% NH,HCO, as the eluent and the protein was recovered by lyophilization. Subunits were obtained from nitrated hCG monomer in the same manner as for preparation of the subunits from the native hormone. 3 Abbreviations used: hCG, human nadotropin; LH, luteinizing (interstitial lating) hormone; TSH, thyroid-stimulating (thyrotopin); TNM, tetranitromethane; dium dodecyl sulfate.

chorionic gocell-stimuhormone SDS, so-



Preparation of tryptic peptides. The nitrated subunits were reduced and the S-carboxamidomethyl derivatives were formed as previously described (5). The S-carboxamidomethyl subunits were dissolved in 1% NH,HCO, to a concentration of 1 mM and hydrolyzed with trypsin (2 g/100 g of substrate protein) at 37°C for 2 h. The hydrolysates were initially fractionated by gel filtration through Sephadex G-50 (superfine) or G-25 (superfine), then by paper electrophoresis, pH 1.8, 90% formic acid-glacial acetic acid-water (55:175:1770, v/v) for 1 hat 115 V/cm. The glycopeptides from hCG-a were separated from the NH,-terminal peptide by countercurrent distribution as previously described (15). Analytical methods. Acid hydrolyses of native and nitrated hCG and its subunits were carried out in the presence of 2.5 mg of phenol to protect tyrosine and hydrolyses of the peptides also contained 2.5 ~1 of thioglycolic acid to protect S-carboxymethylcysteine. Because 3-nitrotyrosine elutes at the position of glucosamine on the 55-cm column in the accelerated system (16), 3-nitrotyrosine was determined separately on this column but eluting with 0.35 M sodium citrate, pH 5.28, as described by Cheng and Pierce (11). Some samples also contained 3-aminotyrosine as described below. This emerges from the 55-cm column eluted with pH 5.28 buffer midway between phenylalanine and 3-nitrotyrosine and has a color yield about 0.8 that of 3-nitrotyrosine. Methionine sulfoxide was determined as described by Neumann et al. (17). Sialic acid was determined by the method of Warren (18). Electrophoresis in 7% polyacrylamide gels at pH 9.5 was carried out by the method of Davis (19) using the buffer system of Liao et al. (20). Gels were stained with 0.1% amido black in 7% acetic acid and destained in 7% acetic acid. Examination of the nitrated p subunit for the presence of crosslinked hCG monomer was by gel electrophoresis in the presence of SDS as described by Weber and Osborn (21). Gels were stained with 0.25% Coomassie Brilliant blue in 45% methanol-lo% acetic acid and destained in 7% acetic acid-5% methanol. Recombinations. Equimolar amounts of the a and 0 subunits were incubated together for 18 h at 37°C in 0.5% NH,HCO,. For analytical samples to be electrophoresed in polyacrylamide gels 40 pg of a and 60 pg of p were incubated in 100 ~1 of buffer, and then lyophilized for 6 h to remove NH,HCO, before being dissolved in the sodium glycinate running buffer. For preparative samples the final protein concentration was 2.5 mg/ml and following incubation these were fractionated by gel filtration through Sephadex G-100 at 4°C with 0.5% NH,HCO, as the eluent. Hormone assays. Biological activity was estimated by the radioligand-receptor binding assay of Catt et al. (22) as modified by Bellisario and Bahl

TETRANITROMETHANE (23). Immunological activity was determined by radioimmunoassay (24). Iodinated hCG was prepared by the procedure of Greenwood et al. (25). Nomenclature. Hormone nitrated with 615 mM TNM is designated l-nitro-hCG and the subunits and peptides derived from it are identified by addending I-nitro-hCG in parentheses, e.g., (Y (l-nitrohCG). Hormone nitrated with 2.0 mM TNM is designated exnitro-hCG and its subunits and peptides are named accordingly. Subunits nitrated in the absence of .the opposite subunit are designated exnitro-cr and ex-nitro-p and peptides derived from them named above. Peptide nomenclature is as described previously (5, 15). Peptides resulting from less extensive hydrolysis than reported in these communications are named as the sum of the peptides previously obtained, e.g., a T-2+3+4.





3 mol of 3-nitrotyrosinelmol of hCG, all of it again present in the (Y subunit. The (Y subunit used for preparation of ex-nitro-o! by reaction with 2.0 mM TNM was that obtained after dissociation of exnitro-hCG. The time course of the reaction was biphasic and is given in Fig. 2. The yield of monomer isolated by gel filtration (Fig. 3a) was 70% and the material contained 4 mol of 3-nitrotyrosine/mol of (Y subunit. In preliminary experiments, nitration of the native (Y subunit under these conditions resulted in only a 30% yield of monomeric material and the biphasic na-


Preparation units

of Nitrated

hCG and Its Sub-

In preliminary experiments it was found that at protein concentrations less than 810 PM the reaction with tetranitromethane did not proceed well. Therefore, in subsequent work a protein concentration of 10 PM was used so as to obtain good reaction rates while minimizing crosslinking (26-28). Monomer yields were 70-85%. Similarly, TNM concentrations of less than 0.1-0.X mM were found to result in poor reaction rates. For the preparation of partially nitrated hCG (1-nitro-hCG) a TNM concentration of 0.15 mM was selected. Exhaustively nitrated material was prepared using a TNM concentration of 2 mM and the reaction was generally complete in 60-90 min. The rate of reaction of hCG with 0.15 mM TNM was linear over a period of 75 min, at which time the reaction was terminated by gel filtration through Sephadex G-25. The nitrated monomer was separated from polymeric material by gel filtration through Sephadex G-100 (Fig. la) and was obtained in 85% yield. This material contained 1 mol of 3-nitrotyrosine/mol of hCG, all of it present in the (Y subunit. The reaction of hCG with 2 mM TNM was complete in 60 min, when the reaction mixture was desalted through Sephadex G-25. The yield of monomer obtained after gel filtration through Sephadex G-100 (Fig. lb) was 75%. The material contained


.0.3 .OP . 0.1 400




FIG. 1. Gel filtration patterns on Sephadex G-100 in 0.5% NH,HCO, of (a) 57 mg of hCG after reaction with 0.15 mM TNM and (b) 105 mg of hCG after reaction with 2.0 mM TNM. The column (2.5 x 190 cm) was run at 4°C at a flow rate of (a) 20 ml/h or (b) 25 ml/h and 20-min fractions were collected. Pooled fractions are indicated by solid bars.

TIME lmin)

2. Time course of the reaction (ex-nitro-hCG) with 2.0 rnxr TNM. FIG.

of 13 mg of a




ture of the reaction could not be observed. The amount of 3nitrotyrosine found in the monomer was the same, however, after



150 200 ml EFFLUENT

FIG. 3. Gel filtration patterns on Sephadex G-100 in 0.5% NHIHCOI of (a) 11 mg of o (ex-nitro-hCG) and (b) 30 mg of 6 (ex-nitro-hCG) after reaction of each with 2.0 mM TNM. The column (1.4 x 200 cm) was run at 4°C at a flow rate of 8 ml/h and 20-min fractions were collected. Pooled fractions are indicated by solid bars. TABLE AMINO Amino


Lysine Histidine A&nine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine 3-Nitrotyrosine” Phenylalanine 3-Aminotyrosine



10.2 3.8 15.2 18.2 17.3 19.6 18.8 31.2 12.0 12.6 14.5 18.4 3.6 5.5 17.7 7.0

(10) (4) (15) (17) (18) (20) (18) (31) (12) (13) (22) (18) (4) (6) (18) (7)

6.0 (6)

a Includes 3-aminotyrosine b Contaminated lo-15%


hCGff (14) 5.8 3.0 3.6 5.7 7.7 8.0 9.5 7.2 4.3 4.7 8.2 7.0 2.8 1.0 3.9 3.6

(6) (3) (3) (6) (8) (8) (9) (7) (4) (5) (10) (7) (3) (1) (4) (4)

3.9 (4)



3.5 0.7 9.5 11.0 9.2 11.6 9.3 24.8 8.4 8.1 9.4 10.1 0.6 3.8 12.6 2.9

(4) (1) (12) (11) (10) (12) (9) (24) (8) (8) (12) (11) (1) (5) (14) (3)

2.1 (2)

value, listed crosslinked


nitration of either (Y (ex-nitro-hCG) or native (Y. The p subunit used for preparation of ex-nitro-p was also obtained by dissociation of ex-nitro-hCG and contained lo-15% of crosslinked hCG monomer as judged by amino acid analysis and SDS gel electrophoresis. Reaction with 2.0 mM TNM was complete in 60 min and resulted in formation of 2.1 mol of 3-nitrotyrosinelmol of hCG@ The yield of monomer obtained by gel filtration (Fig. 3b) was 85%. The amino acid compositions of the nitrated hCG and subunits are given in Table I. The significant differences seen are in the values for tyrosine and 3nitrotyrosine. The differences in the half cystine and arginine values are methodological and are not surprising under hydrolysis conditions employed. Control acid hydrolyses in the presence of bovine serum albumin did not result in destruction of S-nitrotyrosine. However, in the presence of hCG, which contains 30% carbohydrate (121, some reduction to 3-aminotyrosine was observed as well as destruction by other unidentified side reactions. As the carbohydrate portion of hCG includes sialic acid, control hydrolyses were carried I

OF NITRATED cdllnitronitrohCG hCG) 9.9 4.2 15.3 17.8 17.7 20.0 19.1 25.4 12.5 12.5 16.0 17.5 3.7 5.5 18.0 5.9 1.0 6.2 0.4

5.1 2.7 3.0 6.0 8.0 7.3 9.3 7.6 4.3 5.0 7.0 8.0 3.0 1.1 3.8 2.9 0.7 4.0 0.2

separately at bottom hCG monomer.

hCG AND ITS SUBUNITS /3(1exdexp(exnitronitronitronitrohCG) hCG hCG) hCG) 4.2 1.2 10.3 11.3 9.3 12.0 9.7 22.5 8.4 8.9 8.3 11.5 1.2 3.7 8.5 2.9 0 2.3

of table.

10.2 4.0 15.7 17.8 17.4 19.3 19.0 34.8 12.5 12.9 14.6 18.2 3.3 5.7 14.6 3.9 2.7 5.9 0.8

6.4 2.5 3.3 5.8 7.6 7.2 9.7 5.8 4.3 4.8 6.6 7.5 2.9 1.1 4.5 1.2 2.5 4.0 0.4

4.6 1.4 10.9 11.0 9.5 11.8 10.1 15.9 7.9 8.1 8.7 11.2 1.3 4.4 12.2 2.8 0.P 2.2 0.1

exnitrocx 5.0 2.5 3.3 6.2 8.0 7.5 9.5 8.2 4.3 4.8 6.8 7.1 2.6 1.0 4.0 0 1.7 3.6 0

exnitro/3 3.9 1.1 12.3 12.0 10.1 12.7 9.7 25.4 8.2 8.2 7.8 10.9 1.0 4.3 11.7 1.0 2.1 2.2 0.6




out in the presence of 0.35 pmol of Nacetylneuraminic acid. This resulted in 35% reduction of 3-nitrotyrosine to 3-aminotyrosine as well as destruction by other unknown pathways. Because the yield of tyrosine is good under the hydrolysis conditions employed, the disappearance of tyrosine was taken as the best measure of the degree of nitration. Preparation of Tryptic Peptides and Location of Nitrated Tyrosyl Residues The (Y subunits obtained from l-nitrohCG and ex-nitro-hCG and the ex-nitro-a and ex-nitro-/3 subunits were reduced, Scarboxamidomethylated, and hydrolyzed with trypsin. The gel filtration patterns of these hydrolysates are given in Figs. 4 and 5. Amino acid compositions of the peptides containing 3nitrotyrosine are given in Table II. In some peptides, particularly the glycopeptide fractions, considerable destruction of 3-nitrotyrosine, including reduction to 3-aminotyrosine, was observed as was the destruction of S-carboxymethylcysteine. Control hydrolyses in the presence of thioglycolic acid, added to peptide hydrolysates to protect S-carboxymethyl-


FIG. 4. Gel filtration patterns on Sephadex G-50 (superfine) in 0.5% NH,HCO, of the tryptic hydrolysates of (a) 7.3 mg of S-carboxamidomethyl (Y (lnitro-hCG) and (b) 13.6 mg of S-carboxamidomethyl ex-nitro-6. The column (1.4 x 200 cm) was run at 4°C at a flow rate of 5.5 ml/h and 25min fractions were collected. Pooled fractions are indicated by solid bars.



I. 0.03; 7 0


&. 002 s


,j.o.o, f.


i 8


FIG. 5. Gel filtration patterns on Sephadex G-25 (superfine) in 0.05 N acetic acid of (a) fraction 1, Fig. 4a, and the tryptic hydrolysates of (bl 6.4 mg of Scarboxamidomethyl a (exnitro-hCG) and (c) 6.4 mg of&carboxamidomethyl ex-nitro-a. The column (1.4 x 200 cm) was run at 4°C at a flow rate of 3.7 ml/h and 30-min fractions were collected. Pooled fractions are indicated by solid bars.

cysteine, resulted in no reduction or destruction of 3nitrotyrosine. Clearly, the presence of 3-nitrotyrosine during acid hydrolysis can lead to various undesirable side reactions. Again, the disappearance of tyrosine was taken as the best measure of the degree of nitration. The (Y subunit derived from 1-nitro-hCG contained 1 residue of 3-nitrotyrosine. Fractionation of the tryptic hydrolysate of a! (1-nitro-hCG) on Sephadex G-50 yielded three fractions, two of which contained 3nitrotyrosine (Fig. 4a). The first fraction, containing most of the nitrated tyrosine, was subjected to gel filtration on Sephadex G-25 (Fig. 5a) followed by countercurrent distribution to separate the glycopeptides Q T-8 and CYT-11 from the NH,-terminal peptide (Y T-l. Analysis of the glycopeptide fraction showed 0.3 residue of 3-nitrotyrosine (as 3-aminotyrosine) and 0.9 residue of tyrosine, indicating that about 1 residue of tyrosine in this fraction had been nitrated. There are two tyrosines in this fraction, positions 88 and 89, but we have been unable to determine whether one or both of these had reacted. Efforts to define their relative amounts of nitration by hydrolysis of (Y T-11 with thermolysin as pre-



(5) (3)





0.68 0






(2) (1)


I 19

0.39 0.37


2.03 1.68 0.25 2.25 3.11 2.86 2.10 1.29 0.68 2.00 0.69 3.65 0.31 0.15 0.34 (2)

(2) (5) (3)

(2) (4) (3) (2)

(2) (3)

c3T-8 + (YT-11







1.39 0.11 0.97 0.42 0.15 2.07 0.40 0.93 0.63 0.11 0.22















0 0.94


1.15 1.01




(1) (1)


loo.47 0.52 0.32

2.06 1.90 0.36 2.12 3.41 2.76 2.43 1.35 0.87 1.79 1.59 3.42 0.57 0.11 0.63 C2)

(2) (5) (3)

(2) (4) (3) (2)

(2) (3)

CCT-8 + air-11



Q Includes I-aminotyrosine value, listed separately at bottom of table. * Yields are based on the amount of each subunit before S-carboxamidomethylation. c Values less than 0.10 residue are given only for tyrosine, 3-nitrotyrosine, and 3-aminotyrosine. d Contaminated 40% with (uT-2+3+4. e Contaminated 40% with 6T-9.



(4) (3)


1.78 1.04



+ 4 + aT-9

- ,rT-3




+ aT 11


2.02 2.21 0.15 2.68 3.74 2.88 3.38 2.03 1.05 2.11 0.95 3.62 0.45 0.24 0.55



0.30 0.93 > 1.01 0.30


Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine S-Carboxymethylcysteine Valine Methionine Isoleucine Leucine Tyrosine 3-Nitrotyrosinen Phenylalanine 3-Aminotyrosine






0.93 0 0.97

2.20 0.13 0.97













1.06 0.99



(1) (1)



1.02 0.20 2.03 2.54 4.99 0.89 2.19 2.18 2.05 2.87 6.57 2.99 0.68 2.51 1.48 0 0.50 (l)

(2) (3) (2) (3) (5) (3) (1) (3) (1)

(2) (2) (5)






0.55 1.84 1.79 0.89 1.30 1.30 3.04

0.85 0.98





(2) (2) (1) (1) (1) (3)

(1) (1)



0.87 2.80


1.21 0.44 1.19 1.02









5 u


c g


viously described (14) were unsuccessful. Peptide (11 T-9 was recovered with (Y T-3+4 after paper electrophoresis .of fraction 3 (Fig. 4a). Analysis showed 15% nitration of Tyr-65. The cx subunit obtained by dissociation of ex-nitro-hCG contained about 3 residues of 3-nitrotyrosine. The tryptic hydrolysate of this material was fractionated by gel filtration through Sephadex G-25; most of the fractions contained 3-nitrotyrosine (Fig. 5b). Comparison of the gel filtration pattern of the tryptic hydrolysate of (Y(exnitro-hCG) with those of (Y (l-nitro-hCG) (Figs. 4a and 5a) and ex-nitro-a (Fig. 5~) shows that although the same trypsin was used in each case, a! (ex-nitro-hCG) underwent more extensive hydrolysis, notably peptide (Y T-11. The glycopeptides were separated from (Y T-l by countercurrent distribution of fraction 1 (Fig. 5b). Amino acid analysis of a! T-8 plus a T-11 showed essentially complete nitration of tyrosines 88 and 89. Peptide cy T-5, which includes Tyr-37, was isolated by paper electrophoresis of fraction 3 and shown by amino acid analysis not to have been nitrated. Because of the evident hydrolysis of (Y T-11 and the isolation in good yield of nonnitrated a! T-5 from fraction 3, the 3-nitrotyrosine-containing material in fractions 2-4 must have derived from the COOH-terminal region of LYT-11. However, no peptides from that sequence were recovered after paper electrophoresis of these fractions. Fully nitrated (Y T-9, containing Tyr-65, was purified by paper electrophoresis of fraction 6 (Fig. 5b). Fully nitrated hCG-a was obtained by nitration of a (ex-nitro-hCG) and the tryptic hydrolysate of this was fractionated by gel filtration through Sephadex G-25 (Fig. 5~). The glycopeptide fraction, LYT-8 plus (Y T-11, was isolated by countercurrent distribution of fraction 1. About 0.5 residue of 3-nitrotyrosine and no free tyrosine was recovered from the acid hydrolysate, indicating complete nitration at positions 88 and 89. Peptides (Y T-5 and (Y T-9 were isolated by paper electrophoresis of fractions 3 and 5, respectively. Their analyses show complete conversion of tyrosines 37 (a T-5) and 65 (a! T-9) to 3-nitrotyrosine.





Nitration of free p subunit resulted in conversion of about 2 residues of tyrosine to 3nitrotyrosine. One of the nitrated tyrosines (position 37) is contained in peptide LYT-3, recovered with (Y T-2 in fraction 3 (Fig. 4b). Analysis of the acid hydrolysate of this fraction showed 0.5 residue of 3nitrotyrosine and no free tyrosine, indicating complete nitration. The other major position of nitration was Tyr-59, contained in /3 T-4. This peptide was recovered by paper electrophoresis of fraction 5 and amino acid analysis indicated about 85% nitration at this position. The remaining tyrosyl residue in hCG-p, position 82, is contained in /3 T-8. This peptide was isolated by paper electrophoresis of fraction 6 and amino acid analysis showed 25% reaction of this tyrosine. The 3-nitrotyrosinecontaining material’in fraction 7 is (Y T-9 derived from crosslinked monomer contaminating the nitrated p subunit. Nitrated Subunit Recombinants: Gel Electrophoresis and Biological and Immunological Activity

Various combinations of native and nitrated hCG (Y and /3 subunits were incubated together in equimolar amounts. The recombination mixtures were then subjected to disc electrophoresis in polyacrylamide gels and gel filtration through Sephadex G-100 to determine the extent of recombination. Disc electrophoresis was also carried out on nitrated intact hormone. Biological activity of nitrated intact and recombinant hormone preparations was determined by radioligand receptor binding assay and immunological competence was determined by radioimmunoassay. The polymeric material (fractions 1, Figs. la and b) was also assayed. Disc gel patterns are shown in Fig. 6 and percentages of recombination and biological and immunological activity are shown in Table III. Incorporation of one 3-nitrotyrosyl residue near the COOH-terminus of hCG-a has little effect on the electrophoretic mobility of hCG (Fig. 6a). The immunological response is decreased 20%, and the biological activity is decreased by half. The subunits obtained from 1-nitro-hCG behaved



FIG. 6. Polyacrylamide gel patterns at pH 9.5 of various combinations of nitrated and native subunits of hCG. Details of the recombination procedure are given in the text. Samples of native and nitrated intact hCG are 100 pg. (a) Samples 1 to 6 are: 1, native hCG; 2, l-nitro-hCG, 3, ex-nitro-hCG, 4, native hCG-cr; 5, native hCG-8; and 6, native hCG-a plus native hCG-p. (b) Samples 1 to 7 are: 1, 1-nitro-hCG, 2, LI (l-nitro-hCG); 3, native hCG-/3; 4, a (I-nitro-hCG) plus native hCG-p; 5, p (l-nitrohCG); 6, native hCG-a; and 7, /3 (l-nitro-hCG) plus native hCG-cu. (c) Samples 1 to 7 are: 1, ex-nitrohCG, 2, LY(ex-nitro-hCG); 3, native hCG-p; 4, (Y(exnitro-hCG) plus native hCG-P; 5, p (ex-nitro-hCG); 6, native hCG-a; and 7, p (ex-nitro-hCG) plus native hCG-e. (d) Samples 1 to 6 are: 1, ex-nitro-a; 2, native HCG-p; 3, ex-nitro-a plus native hCG-/3; 4, ex-nitro-p; 5, native hCG-a; and 6, ex-nitro-p plus native hCGu.

in an anomalous manner for which we have no explanation. Amino acid analysis of the (Ysubunit showed that the only significant change was the conversion of 1 tyrosyl residue to 3-nitrotyrosine (Table I). The gel pattern of (Y (l-nitro-hCG) was considerably altered, however, and while it is not apparent from the gel patterns (Fig. 6b), its ability to recombine with native hCG-/3 was considerably impaired. This lowered capacity for recombination was not observed with (Y (ex-nitro-hCG).



The biological activity of the a! (l-nitrohCG) native j3 recombinant was 75% that of 1-nitro-hCG. The j3 subunit from l-nitro-hCG was, by all criteria, unmodified. Nevertheless, its ability to recombine with native hCG-a! was also impaired and the recombinant obtained had very low biological activity. In contrast, /3 (ex-nitro-hCG) recombined with hCG-a equally well as did native hCG-/3 and this recombinant had full activity. Exhaustive nitration of hCG resulted in conversion of 3 tyrosines in the (Y subunit to 3-nitrotyrosine (positions 65, 88, and 89). The gel pattern of this hCG was not significantly altered but the mobility was increased (Fig. 6a). The immunological competence of this derivative is about half that of native hCG and the biological activity was only 25%. The banding pattern of (Y (ex-nitro-hCG) is quite different from that of the native (Ysubunit and, although it is unmodified by the criterion of amino acid analysis, the gel pattern of p (exnitro-hCG) also differs from that of native hCG+ (Fig. 6~). Both subunits recombine well with their complementary native subunits; cy (ex-nitro-hCG) to give recombinant hormone with half the activity of exnitro-hCG and p (ex-nitro-hCG) to yield hormone with full native activity (Table III). Exhaustive nitration of the free subunits converts all four tyrosines in hCG-a and two of three in hCG-p to S-nitrotyrosine; the third p-tyrosine is partially nitrated. The gel patterns of each subunit are altered and neither recombines well with the complementing subunit (Fig. 6d). The biological activity of the ex-nitro-onative p recombinant is only 10% of native, while the recombinant of ex-nitro-/3 and native Q! has two-thirds of the native activity (Table III). The polymeric material obtained after either partial or exhaustive nitration of hCG retains about 30% of the native immunological competence but almost none of the biological activity. Crosslinked monomer present in the j3 subunit preparation obtained from ex-nitro-hCG was biologically inactive.







III hCG AND SUBUNIT RECOMBINANTS Radioligand-recepRadioimmunoastor binding” sayGl 1oob 1oob 46 81 25 56 1OOC ndd 12 nd 35 nd 100 nd 13 nd 66 nd 8.5 nd 2.5 36 3.0 30


-Mole’% recombination

Native hCG I-Nitro-hCG Ex-nitro-hCG Native a plus native p Native a plus P(l-nitro-hCG) a(l-Nitro-hCG) plus native /3 Native a plus p(ex-nitro-hCG) a(Ex-nitro-hCG) plus native ,6 Native a plus ex-nitro-p Ex-nitro-a plus native 6 Polymer (I-nitro-hCG) Polymer (ex-nitro-hCG) /3(Ex-nitro-hCG)

72 60 33 77 72 30 35

The reaction of tetranitromethane with human chorionic gonadotropin.

ARCHIVES OF BIOCHEMISTRY AND The Reaction BIOPHYSICS of Biochemistry (1976) of Tetranitromethane Gonadotropin1 ROBERT Department 175, 209-22...
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