Charge properties of human pituitary and amniotic fluid prolactins.

The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The "large" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.     

Ovine luteinizing hormone. I. Effects of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone

Chromatofocusing was used to separate and characterize the isohormones of ovine luteinizing hormone (oLH) in the pituitaries of rams, wethers, and wethers receiving Silastic implants containing 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2), or DHT plus E2. Extracts of anterior pituitaries were prepared by homogenization and centrifugation at 100,000 X g. Castration reduced the amount of oLH in the pituitary, even though peripheral levels were elevated. Pituitary oLH concentrations in wethers were further reduced by all three steroid treatments. When subjected to chromatofocusing on pH 10.5 to 7.0 gradients, pituitary extracts yielded eight peaks of immunoreactive oLH, which eluted with apparent isoelectric points of greater than 9.8, 9.26, 9.14, 9.07, 8.98, 8.91, and less than or equal to 7.0. These isohormones were designated A-G and Z, respectively. In rams, isohormones F and G were the predominant species, representing approximately equal to 57% of the immunoreactive oLH recovered from the column. Castration resulted in a subtle shift toward more basic isohormones. DHT administration caused an increase in the relative amount of isohormone A, whereas E2 treatment resulted in an increase in isohormone Z. DHT and E2 in combination produced increases in the relative amounts of both isohormones A and Z. All eight oLH isohormones were active in an in vitro LH bioassay and exhibited biological-to-immunological-assay (B/I) ratios in the ram ranging from 0.4 to 2.8. Isohormone F exhibited the highest B/I ratio in all the treatment groups. Similarly, isohormone F was clearly the predominant biologically active form of oLH in all groups. These results demonstrate that at least eight immunoreactive and biologically active forms of pituitary oLH can be separated by using chromatofocusing; the pattern of oLH isohormones is markedly different from that in the rat; castration has a minimal effect on the pattern of oLH isohormones in pituitary extracts; and exogenous gonadal steroid administration reduces the amount of oLH in the pituitary and changes the pattern of oLH isohormones, resulting in a higher percentage of less biologically active forms.     

Charge microheterogeneity of ovine follicle-stimulating hormone in rams and steroid-treated wethers

Chromatofocusing was utilized to separate the isohormones of ovine follicle-stimulating hormone (oFSH) in pituitary extracts from rams, wethers, and wethers receiving Silastic implants of dihydrotestosterone (DHT), 17 beta-estradiol (E2), or both DHT and E2. Pituitary extracts were prepared by homogenization and centrifugation at 100,000 x g. Extracts of ram pituitaries yielded at least nine species (isohormones) of immunoreactive oFSH having apparent isoelectric points (pIs) of greater than 7.40, 6.74, 6.52, 5.76, 5.20, 4.74, 4.44, 4.10, and less than 4.00. These isohormones were designated by the letters A-H and Z, respectively. Eighty-four percent of the immunoreactive ram oFSH recovered from the chromatofocusing column was very acidic in nature, having apparent pIs less than 5.30. The majority of the immunoreactive oFSH was focused in an area different from that of most of the immunoreactive ovine luteinizing hormone (oLH) and ovine thyroid-stimulating hormone (oTSH). Pituitary extracts from control and steroid-treated wethers also contained these nine oFSH isohormones, but significant differences were noted in the relative distribution among the isohormones. Castration resulted in a 4-fold increase in isohormone B and a concomitant reduction in isohormones E and H. DHT administration returned these levels to the values observed in the ram, whereas E2 administration produced a significant 2-fold increase in the most acidic form (isohormone Z). The implant combination produced isohormone profiles comparable to that of E2 alone. Neuraminidase treatment in vitro of both crude pituitary extracts or highly purified iodinated oFSH abolished the most acidic form and caused a marked shift in the isohormone pattern to more basic species. These results demonstrate that 1) at least nine isohormones of oFSH can be separated reproducibly from the male ovine pituitary by chromatofocusing, 2) the majority of FSH in the pituitary exists in acidic form, 3) castration and steroid administration alter the distribution of oFSH in the pituitary among its isohormones, and 4) at least a portion of oFSH-charge microheterogeneity appears to be due to the presence of sialic acid residues on the molecule.     

Characterization of equine luteinizing hormone by chromatofocusing

Three equine luteinizing hormone (LH) preparations (eLH-A, -B, and -C) recently have been isolated in our laboratory and were shown to differ in average basicity (eLH-A greater than -B greater than -C). The present study further characterizes these preparations by chromatofocusing. Each of these preparations are comprised of a family of isohormones, with 5 major immunoreactive peaks in the pH range of 7 to 4 (approx. pIs = 6.6, 6.1, 5.7, 5.2, and 4.8), with varying amounts of material eluting to either side of the pH gradient. Although similar isoforms are seen in all three LH preparations, the relative proportions of different isoforms vary in a manner reflecting the average charge properties of eLH-A, -B, and -C. While eLH-A contains predominantly basic forms, eLH-C consists largely of acidic material, and eLH-B is composed mostly of isohormones with pIs intermediate to eLH-A and -C. Chromatofocusing of a crude extract from a single horse pituitary gland revealed isohormone peaks corresponding to those found in the highly purified LH preparations. Peak fractions of the various isoforms were used to generate a variety of activity ratios (LH bioactivity:LH radioimmunoassay (RIA), LH radioreceptorassay (RRA):LH RIA, LH bioactivity:LH RRA, follicle-stimulating hormone (FSH) RRA:LH RIA, and FSH RRA:LH RRA activity ratios). The LH bioactivity:LH receptor binding potency ratio showed a linear increase with increasing isohormone acidity (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the microheterogeneity of anterior pituitary follicle-stimulating hormone in the female rat. Isoelectric focusing pattern throughout the estrous cycle

Anterior pituitary (AP) glands were removed from adult female rats at different times throughout the estrous cycle, and the isohormones of follicle-stimulating hormone (FSH) present within them were separated by isoelectric focusing in polyacrylamide gels (PAGE-IEF; pH range 3.0-8.0). Gel eluents were analyzed for FSH content by radioimmunoassay (RIA) and radioreceptor assay (RRA). All AP samples exhibited several peaks of FSH immunoactivity within a pH range of 6.2-4.0; the major peak constantly exhibited an isoelectric point (pI) of 4.9-4.5. To quantify differences in the IEF pattern of AP-FSH between the pituitaries collected during the different days of the cycle, each PAGE-IEF profile was divided into 7 regions (pI 7.0-6.3 = Area 1, 6.2-5.5 = Area 2, 5.4-5.0 = Area 3, 4.9-4.5 = Area 4, 4.4-4.0 = Area 5, 3.9-3.5 = Area 6, and less than 3.5 = Area 7), and the amount of FSH present within each was determined. In all APs collected at 0900 h of diestrus 1 (D1) and 2 (D2), proestrus (P), and estrus (E); at 1300 h of D1, D2 and E; at 2200 h of P; and at 0200 h of E, the majority of FSH immunoactivity (37-55% of total FSH on gel) focused within Area 4, whereas Areas 2 and 3 contained minor amounts of FSH activity (11-26% and 14-24%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

A three-step method for isolating a few to several hundred milligrams of protein

An operationally simple general protein isolation method was devised from three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with pI values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homogeneous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70--80%.     

Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse

Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms were found to contain an equivalent amount of hexose (9.0-9.2%). Isohormones eLH-B and eLH-C, however, possess more sialic acid than eLH-A (6.6-6.7%, vs. 4.5%). The eLH-A and eLH-B preparations contain a similar amount of hexosamine, which is slightly lower than the amount of eLH-C (8.8-9.1% vs. 11.2%). No differences were noted between the isohormones by rat Leydig cell LH bioassay, equine testis LH radioreceptor assay (RRA) or calf testis follicle-stimulating hormone (FSH) RRA. Slight, but nonsignificant, variations were noted between preparations in an eLH radioimmunoassay (RIA). Although chemical variations were detected between the eLH isoforms, no significant differences were observed in in vitro biological and immunological activities. The differences detected in sialic acid content raises the possibility that differences in in vivo clearance rates may exist.     

A sensitive method for detection of circulating forms of human growth hormone by immunoautoradiography

Several different forms of hGH have been isolated from the pituitary, but little is known about modifications of synthesized forms that may exist in the circulation. We used a combination of serum immunoextraction with gel electrophoresis and electrophoretic blotting to detect circulating hGH isohormones. Three different components were identified, with the predominant form being 22,000 MW isomer. The method is sensitive to nanogram quantities of hGH and is applicable to the screening of human serum for the presence of variants of hGH.     

Isolation of Rat Pituitary Prolactin Isohormones Differing in Charge,Size, and Specific Immunological Activity

A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimnunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay.

A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones.

In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immuno-potency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis.

Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone.     

Influence of bilateral cryptorchidism on rat pituitary luteinizing hormone charge microheterogeneity

Chromatofocusing was used to characterize the isohormones of rat luteinizing hormone (rLH) in extracts of pituitaries from intact and bilaterally cryptorchid animals. Pituitary extracts contained at least seven isoelectric variants (isohormones) or rLH: one eluted in the column void volume (Isohormone, I, pI greater than 9.8), five exhibited apparent pI's in the range of 9.25 to 8.97 (designated as Isohormones II-VI) and one bound to the column but eluted with 1.0 M NaCl (Isohormone VII, pI less than 7.0). In both intact and cryptorchid rats, a large percentage of the rLH was present as Isohormone I. Furthermore, Isohormone I was present in greater absolute and relative amounts in cryptorchid rats. All seven rLH isohormones exhibited activity in an in vitro bioassay and the biological to immunological assay (B:I) ratios decreased with their apparent pI's but were not significantly affected by cryptorchidism. When the data were expressed as the product of the amount of rLH and the B:I ratio (termed Bio-Index), Isohormone I was the predominant form of rLH in both cryptorchid and intact rats, with the Bio-Index of Isohormone I being significantly greater for cryptorchid rats. These results suggest that cryptorchidism alters the pattern of rLH isohormones in the pituitary, yielding a greater percentage of the most basic rLH isohormone, which also has the highest B:I ratio. Thus, cryptorchidism significantly alters the qualitative pattern as well as the quantity of rLH in the pituitary.     

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.     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

Gel electrophoretic procedures potentially applicable to the isolation of human growth hormone from a transformed bacterial source at the gram-preparative scale

A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoretic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM larger than 0.127) at pH 7.6, 0 degrees C, of the bacterial contaminants relative to hGH. This allows one to stack the contaminants at a protein load of 31.5 mg/cm2 of gel, using a "non-restrictive" gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously. Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack ("re-stacking") and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0 degrees C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %CBis, at a protein load of 2.5 mg/cm2 of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm2 gel surface area. The yield of hGH was 90% and its purity ranged from 84-92%.     

Influence of D Genome on the Esterase Isozymes in Wheat

Esterase isozyme zymograms of endosperms at milk stage were analyzed by polyacrylamide vertical slab gel electrophoresis. It was shown that Aegilops squarrosa, Ae. ventricosa and Ae. crassa with D genomes all had E1-1 band with the same migration rate. In zymograms, the two bands E1-1, E1-2 of common wheat (AABBDD) and emmer wheat (AABB) were actually each compossed of two closely adjacent bands. Zone E3 of the two species had five bands each. There were no difference in migration rate of these bands between the two species, but the activities of these enzymes were different and they showed regular change. Zymograms of the hybrids of reciprocal crosses between emmer and common wheat are mainly tended to maternal types and it is representing the dosage effect of D genome.     

Gel Electrophoretic Procedures Potentially Applicable to the Isolation of Human Growth Hormone from A Transformed Bacterial Source at the Gram-Preparative Scale

A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoret ic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM² larger than 0.127) at pH 7.6, 0°C, of the bacterial contaminants relative to hGH. This allows one to stack ³ the contaminants at a protein load of 31.5 mg/cm² of gel, using a “non-restrictive” gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously⁴ Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack (“re-stacking”) and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0°C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %C_. ², at a protein load of 2.5 mg/cm² of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm² gel surface area. The yield of hGH was 90% and its purity ranged from 84–92%.     

Resolution by high-pressure liquid chromatography and partial characterization of multiple forms of cytochrome P-450 from hepatic microsomes of phenobarbital-treated rats

Seven cytochromes P-450 (A, B, C, D, E1, E2 and F) were isolated from hepatic microsomes of phenobarbital-induced rats by a modification of the procedure of Guengerich and Martin [Arch. Biochem. Biophys. (1980) 205, 365-379]. The modification consisted of replacing DEAE-cellulose column by two DEAE-Sepharose CL-6B columns connected in tandem, changing the elution scheme and monitoring the resulting fractions by high-pressure liquid chromatography (HPLC). Cytochrome P-450 forms D, E1, E2 and F having molecular masses of 52.5 kDa, 52.5 kDa, 53.3 kDa and 53.2 kDa, respectively were resolved from the major form of cytochrome P-450 'peak B2' of Guengerich and Martin (above reference). These four cytochromes P-450 were immunologically identical by Ouchterlony double-diffusion analysis. Slight but significant differences were evident in the partial peptide digest maps of these four cytochromes P-450 and catalytic properties of these four forms, though qualitatively similar, demonstrated distinct quantitative differences. Furthermore, HPLC retention times of these four cytochrome P-450s were quite different. Cytochrome P-450 forms A, B and C were distinctly different from each other and from the forms D, E1, E2 and F in the following respects: partial peptide digest maps, catalytic activities, and HPLC retention times. The present results show that cytochromes P-450 considered homogeneous by sodium dodecyl sulfate/polyacrylamide gel electrophoresis may be heterogeneous and contain multiple forms of cytochromes P-450 with different net charges but similar molecular-masses. These studies also demonstrate the capability of HPLC in providing a simple and effective tool for monitoring the separation of cytochromes P-450 showing charge heterogeneity.     

Bioactivity of human growth hormone in urine from acromegalic patients

Bioactivity of hGH in urine from five acromegalic patients was determined by Nb2 bioassay and IM-9 receptor modulation assay (RMA). Urine samples were concentrated by immunoadsorbent chromatography, dialysis and lyophilization. The ratio of the bioactivity by Im-9 RMA and the immunoactivity by RIA was between 0.81 and 1.24 (1.01 +/- 0.19, mean +/- SD). The ratio of the bioactivity by Nb2 bioassay and the immunoactivity ranged from 0.45 to 0.60 (1.37 +/- 0.85). Gel chromatography of hGH in urine samples measured by sensitive sandwich enzyme immunoassay showed that more than 97% of hGH activity was the 22K form. Urinary hGH from acromegalic patients was bioactive in Nb2 bioassay and IM-9 RMA and the bioactivity showed a close correlation with the immunoactivity. The major immunoactive form of hGH (22K) seems to correspond to the bioactivity.     

Rotein Composition Cf Flasmin Preparation “Hcmclysin”

A commercial preparation of human plasmin (Homolysin), capable of catalyzing the transformation of human growth hormone (hGH) into biologically activated species, was analyzed by electrophoresis and electrofocusing on polyacrylamide gel. Each major component of the preparation was characterized with regard to molecular size (retardation coefficient, K_R). molecular net charge (y-intercept on the Ferguson plot, Y₀), apparent isoelectric point (pi') and enzyme activity. The multiple components of Homolysln revealed by staining corresponded to various aggregation states of plasmin and exhibited full serine protease activity. Pclyacrylamlde gel electrophoresis of Homolysln in the presence of sodium dodecylsulfate (SDS) yielded 2 sub units which corresponded in molecular weight to the known plasmin subunits.     

Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay.

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.     

Ovine luteinizing hormone. III. Relationships between the charge heterogeneity of partially purified subunits and the native hormone

Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 X g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed: six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI greater than 9.8), and one was bound to the column (apparent pI less than or equal to 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH alpha, and oLH beta were present at concentrations of 0.907 +/- 0.127, 0.089 +/- 0.020, and 0.010 +/- 0.023 microgram/mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.19 and approximately equal to 0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH alpha and oLH beta) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH alpha in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (approximately equal to 37%) of acidic components when chromatofocused, while more than 97% of purified oLH alpha focused as basic forms having pIs greater than 8.9. When uncombined oLH beta in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI less than or equal to 7.0); purified oLH beta displayed a nearly identical pattern. These results suggest that native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts.(ABSTRACT TRUNCATED AT 250 WORDS)