Blocking leptin action one week after weaning reverts most of the programming caused by neonatal hyperleptinemia in the adult rat.

Hyperleptinemia during lactation programs for higher serum leptin in 30-day-old and adult rats, associated with metabolic changes. Here we evaluated the inhibition of serum leptin at 29 and 30 days on the metabolic phenotype of rats programmed with leptin during lactation. Pups from Wistar rats were saline-injected or leptin-injected from postnatal day 1 to day 10. At 29 and 30 days old, animals were injected with anti-leptin antibody (LA and CA) or saline (LS and CS). In adult animals, higher visceral (+53%) and total fat mass (+33%), hyperleptinemia (+67%), hypertriglyceridemia (+47%), and hypoadiponectinemia (-44%) observed in LS group compared to CS were prevented by immunoneutralization of leptin, since LA group had those parameters values similar to CS group. However, immunoblockade of leptin in normal animals led to the same metabolic changes seen in leptin-treated animals, in addition to lower serum adiponectin (-77% vs. CS) and higher insulin resistance index (+37%). Liver sirtuin1 (SIRT1) was higher (+41%) only in LA group, suggesting a role for SIRT1 in the prevention of leptin programming. Hypothalamic OBR was lower and SOCS3 higher in LS group and these changes were normalized in LA group. In conclusion, blocking leptin action one week after weaning seems to revert most of the alterations observed in rats programmed by neonatal hyperleptinemia. Higher liver SIRT1 expression may be one of the mechanisms involved, leading to a better glucose and lipid metabolism. Our data suggest that the lack or the excess of leptin programs an adverse metabolic phenotype in adulthood.

Maternal prolactin inhibition during lactation is associated to renal dysfunction in their adult rat offspring.

The renal function of rats whose mothers had hypoprolactinemia at the end of lactation was evaluated during development. Lactating Wistar rats were treated with bromocriptine (BRO, 1 mg twice a day, s.c.) or saline on days 19, 20, and 21 of lactation, and their male offspring were followed from weaning until 180 days old. 1 rat from each of the 12 litters/group was evaluated at 2 time points (90 and 180 days). Body and kidney weights, sodium, potassium, and creatinine were measured. Values were considered significant when p<0.05. Adult BRO-treated offspring presented higher body weight (+10%), lower relative renal weight at 90 and 180 days (-9.2% and -15.7%, respectively), glomerulosclerosis, and peritubular fibrosis. At 90 and 180 days, creatinine clearance was lower (-32% and -30%, respectively), whereas serum potassium was higher (+19% and +29%, respectively), but there were no changes in serum sodium. At 180 days, higher proteinuria (+36%) and serum creatinine levels (+20%) were detected. Our data suggest that prolactin inhibition during late lactation programs renal function damage in adult offspring that develops gradually, first affecting the creatinine clearance and potassium serum levels with further development of hyperproteinuria and higher serum creatinine, without affecting sodium. Thus, precocious weaning programs some components of the metabolic syndrome, which can be a risk factor for further development of kidney disease.

Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice.

Antigens and infectious agents that stimulate interferon alpha(IFN-alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN-alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells.

Three-dimensional structure of recombinant human interferon-gamma.

The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.

Early maternal hyperleptinemia programs adipogenic phenotype in rats.

We have previously reported on the treatment of maternal rats with leptin during the three last days of lactation program for overweight and leptin hypothalamic resistance in the offspring. Here we have investigated whether treatment of maternal rats with leptin in the first ten days of lactation can program metabolic dysfunctions on the adult offspring. Lactating rats were divided into 2 groups: rats (LEP) injected with recombinant mouse leptin (8 microg/100 g/body weight, daily during the first 10 days of lactation) and control group (C) that received the same volume of saline. After weaning, all pups had free access to normal diet, their body weight and food intake were monitored at 4 days interval until 180 days, when they were tested for food intake and response to either leptin (0.5 mg/kg body weight, ip) or saline. The offspring from leptin-treated mothers gained more weight from day 69 onward and had higher food intake from day 145 onward, higher amount of visceral adipose tissue (57%), higher serum glucose (10%), and higher serum leptin (135%) at 180 days compared to control group. The food intake was not reduced as expected after acute injection of leptin in these animals, suggesting resistance to the anorexigenic effect of leptin. We conclude that maternal hyperleptinemia in early lactation programed higher food intake, body weight gain due to higher total and visceral fat mass, and resistance to anorexigenic effect of leptin in the adult offspring even when this hyperleptinemia occurred at the beginning of lactation.

Zinc mediated dimer of human interferon-alpha 2b revealed by X-ray crystallography.

BACKGROUND: The human alpha-interferon (huIFN-alpha) family displays broad spectrum antiviral, antiproliferative and immunomodulatory activities on a variety of cell types. The diverse biological activities of the IFN-alpha's are conveyed to cells through specific interactions with cell-surface receptors. Despite considerable effort, no crystal structure of a member of this family has yet been reported, because the quality of the protein crystals have been unsuitable for crystallographic studies. Until now, structural models of the IFN-alpha's have been based on the structure of murine IFN-beta (muIFN-beta). These models are likely to be inaccurate, as the amino acid sequence of muIFN-beta differs significantly from the IFN-alpha's at proposed receptor-binding sites. Structural information on a huIFN-alpha subtype would provide an improved basis for modeling the structures of the entire IFN-alpha family. RESULTS: The crystal structure of recombinant human interferon-alpha 2b (huIFN-alpha 2b) has been determined at 2.9 A resolution. HuIFN-alpha 2b exists in the crystal as a noncovalent dimer, which associates in a novel manner. Unlike other structurally characterized cytokines, extensive interactions in the dimer interface are mediated by a zinc ion (Zn2+). The overall fold of huIFN-alpha 2b is most similar to the structure of muIFN-beta. Unique to huIFN-alpha 2b is a 3(10) helix in the AB loop which is held to the core of the molecule by a disulfide bond. CONCLUSIONS: The structure of huIFN-alpha 2b provides an accurate model for analysis of the > 15 related type 1 interferon molecules. HuIFN-alpha 2b displays considerable structural similarity with muIFN-beta, interleukin-10 and interferon-gamma, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-alpha 2b dimer.

Evaluation of the antitumor activity of recombinant human gamma-interferon employing human melanoma xenografts in athymic nude mice.

Three human melanoma cell lines (G-361, HT-144, and SK-MEL-3) that were highly sensitive to growth inhibition in vitro by recombinant human interferon (rHuIFN-gamma) were adapted to grow as s.c. xenografts in athymic nude mice. Take rates were greater than 90% for all three tumors, with in vivo doubling times of 15, 4, and 15 days for G-361, HT-144, and SK-MEL-3, respectively. Commencing 3 days posttumor implantation mice were treated with daily s.c. injections of rHuIFN-gamma for 30 days over a dose range of 2.4-326 megaunits/mouse/day at a site distinct from the tumor implant. Tumors were measured twice weekly and mice were observed daily for deaths and morbidity until day 60 postimplantation. No apparent antitumor activity was observed in either the G-361 or HT-144 tumors at any dose despite the achievement of high rHuIFN-gamma blood levels. Intralesional treatment of the HT-144 xenograft with rHuIFN-gamma at 240 megaunits/mouse/day did not significantly retard tumor growth or increase lifespan. However, the SK-MEL-3 tumor showed a significant response at the 326-megaunit dose as noted by a tumor growth delay of 11.8 days in treated versus control animals and by an increased number of 60-day survivors. The tumor growth suppression appeared to be greater during the treatment period than during the subsequent observation period. Other experiments employing 326, 530, and 860 megaunits rHuIFN-gamma/mouse/day in mice bearing the SK-MEL-3 tumor demonstrated tumor growth delays of 4.2, 4.8, and 19.8 days, respectively, suggesting a dose response. These data support the conclusions that (a) in vitro antiproliferative activity of rHuIFN-gamma is not necessarily predictive of in vivo efficacy; and (b) relatively high doses of rHuIFN-gamma appear to be required for demonstrating an in vivo antitumor effect in this model.

Specific immunosuppressive effects of constant infusion of 2'-deoxycoformycin.

The effect of continuous infusion into C57BL/6J mice of 2'-deoxycoformycin (DCF), a tight-binding inhibitor of adenosine deaminase, on the biological function of bone marrow stem cells and T- and B-lymphocytes was evaluated. Greater than 85% inhibition of adenosine deaminase in erythrocytes, thymus, and bone marrow was noted after DCF infusion at 0.4 mg per kg body weight per day, while lesser extents of inhibition were characteristic of spleen and lymph nodes. The reconstitution of lethally irradiated C57BL/6J mice with bone marrow cells from DCF- and 0.9% NaCl infused mice of the same strain was compared. The two groups of animals were virtually identical with respect to (a) the number of spleen colony-forming units, (b) the response of splenic lymphocytes to both B- and T-cell mitogens, (c) hematological analysis of peripheral blood elements, and (d) survival time, thus strongly supporting the lack of effect of DCF infusion on the capacity of stem cells to differentiate. In contradistinction, DCF infusion was highly lymphocytotoxic as noted by the severe necrosis in both B- and T-cell regions in lymph nodes and spleen and by the dramatic weight reduction in spleen and thymus. Histopathology of other tissues including bone marrow was normal except for the occurrence of hepatitis. A striking decrease in blastogenesis induced by the mitogens concanavalin A, phytohemagglutinin, and Escherichia coli lipopolysaccharides was also observed after DCF infusion. Consistent with these data, in vitro incubation of bone marrow cells with DCF did not impair the number of spleen colony-forming units produced in lethally irradiated mice. These data suggest a potential use for adenosine deaminase inhibitors in the prevention of graft-versus-host disease in hematopoietic transplantation.

In vitro assessment of the effects of granulocyte-macrophage colony-stimulating factor on primary human tumors and derived lines.

One hundred eighty-nine human tumor specimens were tested in a human tumor cloning assay to determine their growth response to human recombinant granulocyte-macrophage colony-stimulating factor. Of these samples 48 were evaluable for response. Growth stimulation to greater than 150% of controls was noted in 1 of 12 lung cancers (8%) and 1 of 14 breast cancers (7%) but in no other instances for an overall rate of 2 of 48 (4.2%). A dose-response effect was not seen with each of the two stimulated samples responding only at the two lowest concentrations tested. In addition, 7 cell lines derived from human tumors were tested using a metabolic CO2 production assay without evidence of growth stimulation. Samples of normal bone marrow displayed the usual dose-dependent stimulation whether grown in agar or assayed metabolically. We conclude that human recombinant granulocyte-macrophage colony-stimulating factor has minimal effect on the growth of the solid tumors tested and that clinical trials to reduce chemotherapy-associated myelo-suppression may proceed without undue concern for enhancement of tumor growth.

Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.

IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.

Purification and properties of a novel recombinant human hybrid interferon, delta-4 alpha 2/alpha 1.

The human interferon (huIFN) delta-4 alpha 2(5-62)/alpha 1(64-166) is a genetically engineered hybrid that consists of residues 5-62 of huIFN alpha 2 and residues 64-166 of huIFN alpha 1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN alpha subtypes. This novel recombinant hybrid was purified from Escherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17,800 and 17,100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7 x 10(7) units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) on various cell lines of different histological origin appeared to be more comparable to that of huIFN alpha 1 than huIFN alpha 2. The data suggest that huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) hybrid may be a useful tool for understanding huIFN structure-function relations.

Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells.

Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.

Recommended guidelines for the treatment of chemotherapy-induced diarrhea.

PURPOSE: Management of chemotherapy-induced diarrhea (CID) has customarily involved symptomatic treatment with opioids in conjunction with supportive care. Alternatively, patients refractory to conventional therapy have been given octreotide, a somatostatin analogue. Although this agent has been effective against CID, no widely accepted treatment guidelines that incorporate its use currently exist. An expert multidisciplinary panel was convened to formulate clinical practice guidelines for the treatment of CID. METHODS: The panel reviewed clinical data on the management of CID reported in the literature and analyzed currently available tools used to assess CID. Expert consensus was applied when published data were insufficient. Panel members also considered the effect of CID on quality of life and the cost-effectiveness and efficacy of different pharmacologic approaches. Effective resolution of CID and decreases in the need for supportive care or hospitalization were considered to be primary goals in the formulation of the guidelines. RESULTS: The panel formulated suggested practice guidelines for the management of CID that detail recommendations for the assessment and evaluation of diarrhea and the sequence and duration of administration of specific pharmacologic agents. CONCLUSION: The consensus of the panel was that standardized assessment and management of diarrhea is required to effectively control CID. The panel agreed that further data from a National Cancer Institute (NCI)-sponsored intergroup trial is required to determine the optimal dosage of octreotide and its cost in the treatment of cancer. The panel also agreed that further clinical research is warranted to address significant questions about the most effective way to assess and treat CID.

Subunits and their interactions.

There is fairly general agreement that myosin isolated from rabbit skeletal muscle has a molecular weight of about 500,000. The higher values that have been reported apparently reflect protein aggregation related to the method of preparation. On the basis of present evidence, the myosin molecule has an elongate helical core of two f subunits (average weight about 215,000) that extend into a globular head region containing three g subunits (average weight about 20,000). Myosin may be dissociated into subunits by a number of methods. In 5 M guanidine, the myosin molecule is dissociated into f and g subunits, while at pH above 10, the g subunits are dissociated from the intact fibrous core of myosin. The dissociation of g subunits at pH 10 is accompanied by the loss of both ATPase activity and actin-binding capacity; however, the exact biological significance of the g subunits is presently uncertain. In preliminary studies, the f subunits appear to contain the sulfhydryl residues currently implicated in myosin ATPase, and there is some indication of allosteric regulation of enzymic activity.

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4.

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.

Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.

Preclinical biology of alpha interferons.

The availability of large quantities of purified recombinant human alpha interferons has permitted an expansion of studies on the preclinical biology of interferon. These purified preparations have definitively established that interferon exhibits pleiotypic effects on cellular function, including antiviral, antiproliferative, and immunomodulatory activities. Thus, interferon can exert therapeutic activity by both direct effects on the growth of the tumor and by modulation of the biologic response of the host. The exact mechanism of action will most probably vary from patient to patient. The variety of biologic activities that interferon displays, as well as the fact that it is highly species-specific, makes it difficult to design preclinical studies that can provide guidance for the clinical application of interferon as an antineoplastic agent. Two models that have provided useful preclinical data on human cell lines or fresh biopsies are reviewed: the human tumor clonogenic assay and human tumor xenografts in immunodeficient mice. These models indicate that interferon is likely to be an effective treatment for a broad range of malignancies, and that the response will be highly dependent on the type of interferon as well as its dose, schedule, and route of administration. Both models have provided evidence that alpha interferon may demonstrate a synergistic or additive interaction with standard chemotherapeutic agents like cyclophosphamide or doxorubicin. The utility of any preclinical model for predicting an individual patient's response to interferon is yet to be established.

Molecular and biologic characterization of recombinant interferon-alpha2b.

Recombinant IFN-alpha2b (Intron A, Schering-Plough Corp, Kenilworth, NJ), derived from Escherichia coli, is currently approved worldwide for the therapy of various cancers and chronic hepatitis B and C. We describe here the purity and identity tests for both physicochemical properties and bioactivity that have been developed for the characterization of highly purified IFN-alpha2b with a specific activity of 2.5 x 10(8) IU/mg protein. The data indicate that a product of high purity can be consistently produced without DNA contamination. The antiviral bioassay chosen to measure potency is based on the ability of IFN-alpha2b to protect human foreskin fibroblast FS-71 cells from the cytopathic effects of encephalomyocarditis virus. Various immunoassays are described that have been used to quantitate the presence of binding and neutralizing antibodies in patients treated with IFN-alpha2b. Overall, the available data indicate a very low incidence of neutralizing antibody formation. We also summarize the current state of knowledge with respect to IFN reference standards for the biologic assay as well as recommendations for the calculation of antibody neutralization titers.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.