Dynamic pattern of estradiol binding to uterine receptors of the rat. Inhibition and stimulation by unsaturated fatty acids

The binding of estradiol to uterine cytosoluble receptors from 24-day-old rats was reduced or potentiated by unsaturated fatty acids (NEFAs), depending on the concentrations of estradiol and unsaturated NEFAs. At estradiol concentrations of up to 1.5 x 10(-8) M, unsaturated NEFAs inhibited estradiol binding to the 8 S cytosol receptor. This inhibition was dose-dependent (10-70%, p less than 0.001) and a function of NEFA unsaturation. Scatchard analysis indicated that unsaturated NEFAs caused a large decrease in receptor affinity for estradiol. Polyunsaturated NEFAs had no apparent effect on estradiol binding at estradiol concentrations of 2-4 x 10(-8) M. At high estradiol concentrations (above 4 x 10(-8) M), estradiol binding was increased 130-250% (p less than 0.01) by polyunsaturated NEFAs. This increased binding was particularly associated with proteins sedimenting at 12.5 S and the 8 S binding was, in fact, reduced. Metabolic studies showed that the reduced binding in the presence of unsaturated fatty acids was correlated with a decrease in reversibly bound estradiol at low estradiol concentrations. The increase in estradiol binding at high estradiol concentrations is the result of a reduction in reversibly bound estradiol and an increase in nonorganic solvent-extractable (water-soluble) estradiol. The amounts of these water-soluble estradiol derivatives depended on both estradiol and unsaturated NEFA concentrations. 70% of the water-soluble estradiol derivatives were trichloroacetic acid-precipitable, suggesting a covalent protein-steroid link. Thus, changes in the hydrophobic fatty acid environment of the uterine cytosol estrogen receptor could modify estrogen-receptor function by altering binding site conformation and/or by inducing changes in estradiol metabolism.     

Plasma sex steroid binding in Chiroptera

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.     

Steroid Binding by Mouse Salivary Proteins

The goals of this study were to determine thesteroid-binding specificity of the mouse salivaryandrogen-binding protein (ABP) family and to ascertainwhether there might be other proteins in mouse saliva capable of binding steroids. The optimalconditions for testosterone binding by mouse salivaryproteins were determined using a small-scalechromatography system to separate bound from unboundsteroid. Testosterone binding appeared to be biphasicbut was directly proportional to saliva concentration,with an optimum temperature of 37 C in the second phase.These results were used to develop a steroid-binding protocol to study the steroid specificity ofsalivary proteins separated by electrophoresis. The ABPfamily bound testosterone and progesterone well andHO-progesterone and DHT to a lesser extent but did not bind either cholesterol or estradiol.Steroid structural comparisons suggest that binding byABP is governed by the A ring of the steroid. Anotherprotein that is not a member of the ABP family bound cholesterol specifically but no protein thatspecifically bound estradiol was observed.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

Post-natal patterns of plasma androgen-binding activity in Djungarian (Phodopus sungorus) and golden (Mesocricetus auratus) hamsters

The binding of sex steroids to plasma proteins was examined in post-natal Djungarian and golden hamsters. With dihydrotestosterone or testosterone as ligands, steady-state polyacrylamide gel electrophoresis revealed 2 androgen binding components in the plasma of young Djungarian hamsters of both sexes. The fast-moving component exhibited a low affinity and high capacity for androgens and corresponded to albumin in stained gels. In contrast, the slow moving component was a beta-globulin with high affinity (Ka = 10(9) M-1) and low capacity for androgens, and was identified as a specific sex steroid-binding protein (SBP; also SHBG). This SBP did not bind oestrogens or corticosteroids and was electrophoretically distinct from corticosteroid-binding globulin (CBG). In addition, this protein did not appear to be of testicular origin because it was present in immature females and in immature males that had been castrated for 8 days. Plasma concentrations of SBP in males as measured by a diethyl-aminoethyl-cellulose binding assay were low at birth, became significantly elevated shortly thereafter when plasma androgen values were also elevated, and subsequently fell to low levels during puberty. These changes follow the same general pattern that has been described for other mammals, including humans, during this period of reproductive development. Although the significance of elevated SBP concentrations during prepuberty has yet to be determined, it appears that the increased concentrations of high-affinity androgen binding in the plasma of Djungarian hamsters plays a role in the asynchronous pubertal development of the testes and accessory organs which occurs in this species. The post-natal SBP pattern in females was similar to that observed in males. Plasma SBP levels were low or undetectable in adults of both sexes. As previously described for adult golden hamsters, the plasma of post-natal male and female golden hamsters lacked a specific SBP: androgen binding in this species is apparently limited to albumin and CBG.     

Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).     

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.     

Conformational changes in rodent and human alpha-fetoprotein: influence of fatty acids

Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Purification,characterization, and synthesis of vitellin from the cabbage butterfly Pieris rapae L.

Vitellin from the cabbage butterfly Pieris rapae L. was purified and characterized by electrophoresis. Vitellin from P. rapae is a phosphorylated glycolipoprotein of 380,000 ± 10,000 molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis. Two subunits with an M_r of 150,000 and 40,000 were obtained from vitellin. The native molecule is thought to be a tetramer composed of two molecules of each of these subunits. The isoelectric point, as determined by isoelectric focusing on polyacrylamide gels, is 6.10. Vitellin and vitellogenin were indistinguishable by immunological methods such as double diffusion and tandem-crossed immunoelectrophoresis. Vitellogenin from the hemolymph and vitellin from the ovary were quantified by rocket immunoelectrophoresis. Vitellogenin and vitellin were first detected in 6-day-old pupae, and their levels increased continuously during ovarian development. Vitellogenin synthesis by the fat body in 4-day-old female pupae could be induced by juvenile hormone I.     

Purification of the sex steroid-binding protein from common carp (Cyprinus carpio) plasma.

1. Sex steroid-binding protein was purified from common carp plasma. 2. Testosterone- and estradiol-binding activity existed at the same fraction eluted from gel Sepharose CL-2B, DEAE-Sephacel, hydroxylapatite and HPLC. 3. The molecular weight of the sex steroid-binding protein was 194,000. 4. At 50% displacement the order in which the steroids displaced [3H]testosterone bound to the binding protein was as follows: androstenedione greater than estradiol-17 beta greater than 11-deoxy-17-hydroxycorticosterone greater than 17 alpha-hydroxyprogesterone greater than progesterone greater than deoxycorticosterone greater than estrone greater than 11-ketotestosterone greater than 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one greater than androstenedione greater than pregnenolone greater than cortisone greater than cortisol.     

Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol.

Sex steroid-binding protein receptor was detected on membranes prepared from human premenopausal endometrium. The binding of sex steroid-binding protein to membranes was specific, saturable, and high affinity. Scatchard analysis showed the presence of two binding sites at different affinities. The addition of estradiol (10(-8) M) did not produce any inhibition of binding; indeed, it resulted in a modification of binding characteristics. The demonstration of sex steroid-binding protein receptor on membranes of human premenopausal endometrium indicates that the expression of receptor on membranes is not an effect of estrogen over stimulation on target tissues. Estradiol could act as a modulating factor of the binding, probably reflecting the sensitivity of tissues to different steroids.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10⁻¹⁰−10⁻⁶M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [¹²⁵I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [¹²⁵I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.     

Sex hormone-binding globulin: Anatomy and physiology of a new regulatory system

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are interwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

Characterization of an androgen binding protein (ABP) in rat testis and epididymis

An androgen binding protein (ABP) was demonstrated in the 105,000 g supernatant of rat testis homogenate after charcoal extraction of endogenous steroids. Testis ABP proved to be identical to an ABP previously described in rat epididymis. It contained saturable high-affinity sites which exhibited binding specificity for dihydrotestosterone (6) and testosterone when measured by polyacrylamide gel electrophoresis or by competitive binding using charcoal adsorption. Binding to ABP was not affected by ribonuclease or neuraminidase but was decreased by the disulfide reducing agent, dithiothreitol and the sulfhydryl reagent, N-ethylmaleimide. Binding was abolished by treatment with proteolytic enzyme. The mean molecular radius of ABP was 2.92 nm as determined by the retardation of electrophoretic mobility in polyacrylamide gels of decreasing pore size. Assuming a partial specific volume of 0.66–0.74 the molecular weight was 86,000–91,000 for a spherical molecule. ABP binding was stable after treating at 45° C for 20 min. but was destroyed at 60° C. Binding was maximal between pH 7.5 and 9.0 and decreased at pH below 7.0.     

Studies on the reaction between human growth hormone and oleic acid using polyacrylamide gel electrophoresis.

Through the work of U. J. Lewis and E. V. Cheever (1967, Endocrinoloyg81, 1338–1348) and U. J. Lewis, E. V. Cheever, and B. K. Seavey (1968, J. Biol. Chem.243, 260–267) it has been known for a number of years that human growth hormone (hGH), and many other proteins, reacts with unsaturated fatty acids to give rise to species with enhanced electrophoretic mobility. In view of the possible importance of this reaction in the genesis of charge isomeric protein artifacts, and for the understanding of hGH as a system of multiple isomers with distinct, and in some cases enhanced, specific activities, the nature of this reaction was investigated further. It was found that (1) the positions of oleic acid and growth hormone on polyacrylamide gel electrophoresis (PAGE) are coincident, indicating that the reaction leads to binding of the fatty acid to the protein: (2) the increment in molecular net charge on growth hormone is proportional to the molar ratio between the reactants, oleic acid and hGH; (3) the binding is noncovalent since it reverses under conditions of isoelectric focusing; (4) the reaction product has a molecular size indistinguishable from that of the reactant, hGH, by the criteria of “quantitative” PAGE (however, the reaction product exhibits an elevated negative molecular net charge in PAGE at pH 10.2); (5) the apparent isoelectric points of the hormone and its reaction products with oleic acid are indistinguishable in isoelectric focusing; (6) the interaction does not seem to involve the carboxyl charges on oleic acid since it is independent of ionic strength; (7) a noncovalent hydrophobic interaction with the protein is indicated since the range of electrophoretic mobilities exhibited by the hGH-oleic acid complex is smaller in the presence of benzyl alcohol in the gel than that exhibited by controls in it absence; (8) the reaction does not seem to involve free radical derivatives of the unsaturated fatty acid since it is not altered when the polyacrylamide gel is in a nonoxidative state; (9) the effect of the reaction with oleic acid on the tryptophan spectrum reflects only nonspecific interaction of the hormone-concomitant tryptophan perturbation.