Absolute on-line molecular mass analysis of basic fibroblast growth factor and its multimers by reversed-phase liquid chromatography with multi-angle laser light scattering detection.

The multi-angle laser light scattering (MALLS) detection method was combined with reversed-phase high-performance liquid chromatography to analyze multimerization of basic fibroblast growth factor (bFGF) formed by oxidation of bFGF with air or with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). This analysis provided the absolute molecular mass and the mean square radius for each eluted protein fraction or each slice of the chromatogram. It was shown that depending on the oxidation conditions, bFGF forms different multimeric forms, from dimers to hexamers. It was found that these multimers have varied conformations of the same molecular mass, but different structure. Molecular mass and size analyses provided molecular conformation of the aggregates: the results indicated the formation of rod-like rigid structures. The MALLS analysis confirmed that, during oxidation, each bFGF monomer bound sequentially to form the extended multimer. The proposed scheme of bFGF oxidation with DTNB revealed that the difference in the aggregate structural forms was probably due either to the presence of covalently bound residues of nitrobenzoic acid in the products of oxidation, or to the participation of sulfhydryl groups in disulfide bond formation.

Quantitation of proteins using HPLC-detector response rather than standard curve comparison.

Modern high performance liquid chromatography (HPLC)-diode array detectors with features such as multiple wavelength monitoring are capable of maintaining a high degree of response reproducibility over extended periods of time. This reproducibility suggests that detector response factors, rather than dilution based standard curves, might be used to measure concentrations of proteins and pharmaceuticals. Four different HPLC methods were used to analyze a single protein and to test the accuracy and precision of measurements using response factors. These results were compared to the accuracy and precision obtained using fitting to a standard curve. Protein solutions were analyzed by HPLC after the concentration was determined by quantitative amino acid analysis. The extinction coefficient at 277 nm of these protein solutions was determined by UV spectroscopy as well as calculated based on the known amino acid composition. The theoretical extinction coefficient calculated by summing the extinction coefficient of the individual amino acids was within 2% of the experimental value. Response factors at 215 and 277 nm were calculated using the peak area produced by the injection of a known amount of protein. When the experimental extinction coefficient was used to calculate the expected HPLC-signal response (peak area = absorbance x duration), the recovery of the protein (accuracy) was 100% if measured at 215 nm and between 90 and 94% when measured at 277 nm. The ruggedness of the recovery was between 2.6 and 4% relative standard deviation, depending on the HPLC-method. It was found that the quantitation was at least as accurate when calculated from the peak area using the response factor as when a standard curve was used.

Approaches to analysis of aggregates and demonstrating mass balance in pharmaceutical protein (basic fibroblast growth factor) formulations.

Denaturation, aggregation, and precipitation, which are common events in protein aging, limit the development of pharmaceutical protein formulations. Using the example of basic fibroblast growth factor (bFGF), we describe a systematic approach for quantitative recovery of soluble and insoluble aggregates in aged samples to achieve mass balance in three analytical methods, UV spectroscopy, size exclusion HPLC (HP-SEC), and reverse phase HPLC. Soluble aggregates were evaluated by UV spectroscopy and HP-SEC; the latter method was modified to include 2 M guanidine hydrochloride (GnHCl) in the mobile phase in order to differentiate and simultaneously analyze native and denatured protein. Insoluble aggregates were first solubilized with GnHCl and then recovered quantitatively with the modified HP-SEC method. Chaotrope treatment did not affect the UV peak absorbance but altered the HPLC profiles. The changes were consistent with dissociation of disulfide-linked multimers to monomers with an intramolecular disulfide linkage. This phenomenon was used for estimation of the monomer-multimer distribution in the untreated aggregated sample. These methods established approaches for quantitative recovery and characterization of bFGF aggregates.

Stability of rhbFGF as determined by UV spectroscopic measurements of turbidity.

Loss of potency of a protein formulation due to precipitation of the protein is a major concern to the pharmaceutical scientist. A simple screening method was developed to study the effect of excipients on protein precipitation. It will not provide accurate stability data but it allows the rejection of excipients that may interfere with the stability of a protein formulation. The method is based on measuring the increase in turbidity at 277 nm by UV-spectroscopy and was sensitive and reproducible enough to obtain data within 15 hr at 30 degrees C or 40 degrees C, which will allow prediction of precipitation behavior that would need with conventional methods 2-3 years. Human recombinant basic fibroblast growth factor (rhbFGF or bFGF) was formulated at various pH-values as well as in the presence of various concentrations of preservatives, surfactants, gelling agent, EDTA, NaCl, sodium sulfate, sucrose, and glycosaminoglycans (GAG). Most excipients increased bFGF aggregation rate when their concentrations were increased. Exceptions were heparin and some of its derivatives, and sodium sulfate; high concentrations of sucrose and sodium chloride suppressed aggregation.

Comparison of clearance and metabolism of infused cholecystokinins 8 and 58 in dogs.

BACKGROUND: Cholecystokinin (CCK) 58 is the predominant molecular form of CCK in canine and human intestine and circulating blood. There is no report on the metabolism and clearance rate of CCK-58. The aim of this study was to compare the in vivo half-life and metabolism of CCK-58 with that of synthetic CCK-8. METHODS: CCK-58 was purified from canine intestine by consecutive high-performance liquid chromatographic (HPLC) and fast protein liquid chromatographic steps. The peptides were given to 12 dogs as an intravenous (IV) bolus injection to determine the half-life of circulating CCK. Six dogs were given CCK-58 or CCK-8 as a constant IV infusion to determine plasma clearance rates and stability in circulating blood. Circulating molecular forms of CCK were determined by radioimmunoassay after extraction of CCK from plasma and characterization by HPLC. RESULTS: The half-life of CCK-58 was 4.4 +/- 0.6 minutes compared with 1.3 +/- 0.1 minutes for CCK-8. Less than 5% of CCK-58 could be detected as smaller forms during constant IV infusion. CONCLUSIONS: The longer half-life of CCK-58 compared with CCK-8 and the minimal conversion into smaller forms during constant IV infusion are consistent with the finding that CCK-58 is not only the major stored form but also the circulating form of CCK after endogenous stimulation in dogs.

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.

Accelerated in vitro degradation of CCK-58 in blood and plasma of patients with acute pancreatitis.

Proteases released into the circulation during acute pancreatitis may hydrolyse circulating peptide hormones leading to altered regulatory functions. Cholecystokinin is a major regulator of postprandial gut function; stimulating pancreatic enzyme secretion, gallbladder contraction and diminishing food intake. Cholecystokinin-58 is the largest and most abundant form of this hormone in acid extracts of human intestine, and major amounts are released into the circulation after feeding. In order to test whether cholecystokinin-58 is degraded more rapidly due to the increased circulating of enzymes, this peptide was added to blood and plasma of patients with acute pancreatitis and incubated for various time intervals. The in vitro half life of cholecystokinin-58 was 10 +/- 1 minutes (mean +/- SE) in plasma and 11 +/- 1 min in blood from patients with acute pancreatitis, about four fold lower than the half life in plasma of healthy volunteers; 45 +/- 5 min. Degradation of cholecystokinin-58 produced immunoreactive forms of cholecystokinin that eluted in the positions of cholecystokinin-8 and cholecystokinin-33/39. We conclude that acute pancreatitis increases the degradation of CCK molecules.

Molecular variants of cholecystokinin after endogenous stimulation in humans: a time study.

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the major form of cholecystokinin in human intestine: CCK-58.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

Action of neurotensin on meal-stimulated gastric acid secretion in the dog.

In six to nine mongrel dogs the effect of graded doses of intravenous neurotensin (188, 375, 750, and 1500 pmol/kg h) on acid secretion basally or stimulated by distention (by isotonic glucose), peptone (0.5, 1, and 4 g%), and pentagastrin was studied. Neurotensin did not affect acid secretion basally, stimulated by distention, or the maximal peptone dose. However, when submaximal doses (0.5 and 1 g%) of peptone were instilled in the stomach, neurotensin stimulated the secretory response to intragastric peptone. This effect was observed in doses of intravenous neurotensin which mimicked circulating neurotensin concentrations after a standard test meal. Thus, neurotensin could be considered a physiologic stimulant of acid secretion when protein is present in the stomach. The mechanism for this action of neurotensin is unknown but could be partly explained by an enhanced release of gastrin. The potentiating effect of neurotensin on peptone-stimulated acid secretion could play a major role in gastric secretory function of the dog.

Structural characterization of canine PYY.

PYY was purified from canine colonic mucosa by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence, amino acid and mass spectral analyses of the purified peptide and its tryptic fragments were consistent with the structure: YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY-amide. Canine PYY(1-36) has the identical sequence as porcine and rat PYY but differs from human PYY at position 3, with Ala instead of Ile, and position 18, with Ser instead of Asn. A smaller form, PYY(3-36), was also purified and characterized. It may differ in its biological activity from the intact peptide and could act as a partial antagonist or agonist of PYY(1-36).

A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36).

A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supernatant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two peptides were consistent with the structures: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(1-36)] and--IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(1-36) differs from porcine PYY only at position 3, with Ile instead of Ala, and position 18, with Asn instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(1-36).

Cholecystokinin-58 is the major molecular form in man, dog and cat but not in pig, beef and rat intestine.

Cholecystokinin (CCK)-58 was found to be the most abundant form in upper small intestinal mucosa of man, dog and cat. However, in pig, beef and rat upper small intestinal mucosa CCK-33/39 and smaller CCK-forms were dominant. The differences in the distribution of the molecular forms of cholecystokinin between these species presumably reflects altered posttranslational processing of procholecystokinin. This may be caused by the different feeding habits of the investigated species. The different forms of cholecystokinin were distributed over the entire length of the mucosa in canine small intestine. The total amount of CCK decreased from the duodenal mucosa towards the colon. In the canine duodenal mucosa, CCK-58 accounted for 85% of the total CCK-like immunoreactivity. The relative amounts of small forms of CCK increased towards the distal jejunum.

Detection of cholecystokinin-58 in human blood by inhibition of degradation.

Although cholecystokinin-58 (CCK-58) is a major molecular form stored in the intestine, it has not yet been shown to be released into the circulation. This report describes in vitro degradation of CCK-58 in human blood and plasma and the molecular forms detected when this degradation is inhibited. After incubation of CCK-58 for 150 min between 20 and 24 degrees C, approximately 60% of immunoreactivity recovered was degraded to smaller immunoreactive forms. Storage of the 150-min incubate at -20 degrees C for 3 days greatly increased the observed degradation to 85%. When CCK-58 was added in vitro to blood, similar degradation occurred. Degradation of CCK-58 could be inhibited by addition of acid. Blood was obtained 1 h after a test meal designed to stimulate CCK release. The pH was lowered during collection and processing of blood and plasma to inhibit in vitro degradation of cholecystokinin. This method permitted the detection of significant amounts of CCK-58 in circulation.

Cholecystokinin-58 is the major circulating form of cholecystokinin in canine blood.

Cholecystokinin-58 (CCK-58) is the largest and most abundant, biologically active form of cholecystokinin in canine intestinal mucosa. Despite the high amounts in mucosa, CCK-58 has not been detected in significant amounts in the circulation. The release of CCK-58 into the peripheral blood in response to an intraduodenal perfusion of sodium oleate (9.0 mmol h-1) was studied in seven conscious dogs. Plasma (50 ml) was obtained before and after endogenous stimulation by a newly developed method that prevents in vitro degradation of large cholecystokinins. The relative abundance of immunoreactive forms of CCK was studied by high pressure liquid chromatography (HPLC) which separated the gastrin and CCK forms. Column eluates were measured with an antibody which recognizes the intact carboxyl terminus of both gastrin and CCK. Cholecystokinin immunoreactivity increased over basal in plasma by 7 fmol/ml after intraduodenal perfusion with sodium oleate. The most abundant form of stimulated cholecystokinin immunoreactivity eluted on HPLC in the position of CCK-58 (63% of total immunoreactivity found). Since CCK-58 is biologically active and is the most abundant circulating form, it should play an important role in the physiology of cholecystokinin.