Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
1.
Pharm Res ; 18(3): 261-6, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11442262

ABSTRACT

PURPOSE: BDNF, a noncovalent homodimer, was modified by covalently attaching polyethylene glycol (PEG) with an average molecular weight of 20kDa to the N-terminal methionine. Stability of modified BDNF (PEG-BDNF) in aqueous solution was compared to BDNF after storage at elevated temperature in the presence and absence of NaCl. METHODS: SDS-PAGE. Light Scattering and Size Exclusion Chromatography were used to assess conformational stability and chemical degradation. In addition, CD spectroscopy was used to follow changes in secondary and tertiary structures upon thermal stress of the protein. RESULTS: NaCl containing formulations are more stable than NaCl-free formulations. In NaCl-free formulations, the main degradation product of BDNF and PEG-BDNF had a molecular weight of monomer that was more chemically degraded than the dimer. Additionally, the degradation of PEG-BDNF occurred at an accelerated rate compared to BDNF in NaCl-free environments. CONCLUSIONS: The addition of NaCl to formulations enhances the shelf-life and conformational stability of both BDNF and PEG-BDNF.


Subject(s)
Brain-Derived Neurotrophic Factor/chemistry , Sodium Chloride/chemistry , Circular Dichroism , Drug Stability , Electrophoresis, Polyacrylamide Gel , Excipients , Kinetics , Molecular Conformation , Molecular Weight , Polyethylene Glycols/chemistry , Protein Conformation , Scattering, Radiation
2.
Protein Sci ; 8(11): 2519-23, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10595558

ABSTRACT

Asn deamidation was monitored in Ala-based octadecapeptides of varying alpha-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala --> Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of alpha-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.


Subject(s)
Asparagine , Peptides/chemistry , Protein Structure, Secondary , Alanine , Amino Acid Sequence , Glycine , Kinetics , Molecular Sequence Data , Structure-Activity Relationship , Thermodynamics
3.
J Chromatogr A ; 798(1-2): 47-54, 1998 Mar 06.
Article in English | MEDLINE | ID: mdl-9542125

ABSTRACT

The complete removal of the fluorescent cysteine derivative 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) from an intact protein has not been demonstrated even after extended treatment with a reducing agent. It has been suggested that this may be due to incomplete denaturation under the conditions employed. We were interested in investigating this phenomenon utilizing small peptides containing individual ABD-labeled cysteine residues. After incubating the fluorescent peptides in the presence of a reductant, it was shown that the ABD label could be completely removed from all of the cysteine-containing peptides investigated. Therefore, delabeling irreversibility is due to residual structure in proteins. Electrospray ionization mass spectrometry (ESI-MS) was used to determine the molecular mass of each peptide after removal of the ABD lavel. The ESI-MS data were consistent with the generation of a free sulfhydryl. The generation of the free sulfhydryl was further substantiated when a delabeled peptide was completely relabeled with ABD-F in the absence of reductant.


Subject(s)
Cysteine/chemistry , Fluorescent Dyes , Oxadiazoles , Peptides/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Dithiothreitol/pharmacology , Hydrogen-Ion Concentration , Mass Spectrometry/methods , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Reducing Agents/pharmacology
4.
Pharm Res ; 15(12): 1822-7, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9892464

ABSTRACT

PURPOSE: Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. METHODS: Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. RESULTS: The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). CONCLUSIONS: PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.


Subject(s)
Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Thrombopoietin/chemistry , Thrombopoietin/metabolism , Alkylation , Anti-Bacterial Agents , Chromatography, High Pressure Liquid , Diketopiperazines , Drug Storage , Humans , Hydrogen-Ion Concentration , Mass Spectrometry , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Mapping , Piperazines , Polyethylene Glycols/analysis , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Temperature , Thrombopoietin/analysis
5.
Pharm Res ; 13(7): 996-1002, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8842035

ABSTRACT

PURPOSE: The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue. METHODS: The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules. RESULTS: Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation. CONCLUSIONS: We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.


Subject(s)
Granulocyte Colony-Stimulating Factor/chemistry , Granulocyte Colony-Stimulating Factor/analogs & derivatives , Polyethylene Glycols , Recombinant Proteins
6.
J Immunol Methods ; 187(1): 151-61, 1995 Nov 16.
Article in English | MEDLINE | ID: mdl-7490451

ABSTRACT

The chelating agent diethylenetriaminepentaacetic acid (DTPA) has been conjugated site-specifically to the N-terminus of recombinant human interleukin-2 (rhIL-2) by reaction with DTPA dianhydride at an initial pH of 6.0, thus demonstrating broader application of the conjugation method previously described for the structurally related cytokine rhG-CSF (Ralph et al., 1995). Purity of the DTPA-rhIL-2 conjugate, isolated by cation-exchange FPLC, and chelation of 111In were revealed by cation-exchange HPLC. Purity of the conjugate as well as chelation of radiometal were also demonstrated by SDS-PAGE and TLC, respectively. The stoichiometric molar ratio of DTPA to protein for the conjugate was approximately 1:1 as determined by TLC and mass spectrometry. Localization of the DTPA moiety was resolved by a peptide mapping procedure. The protein retained > 95% secondary structure (alpha helicity) following the conjugation. Addition of metal induced an approximate 22% loss of secondary structure for the conjugate. The in vitro biological activity of the protein was unaffected by the conjugated DTPA, even with chelated metal. Pharmacokinetic analysis of DTPA-conjugated cytokines, following chelated 111In, showed clearance and pharmacokinetic parameter values comparable to those of the corresponding unmodified cytokine. DTPA-conjugated cytokines may prove useful in cytokine research, and furthermore may represent a novel class of molecules for imaging, diagnosing, and/or treatment of malignancies where the cytokine receptor is overexpressed.


Subject(s)
Chelating Agents/metabolism , Interleukin-2/metabolism , Interleukin-2/pharmacokinetics , Pentetic Acid/metabolism , Amino Acid Sequence , Chromatography, High Pressure Liquid , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Indium Radioisotopes , Interleukin-2/chemistry , Molecular Sequence Data , Pentetic Acid/chemistry , Protein Binding , Receptors, Interleukin-2/analysis
7.
Biochemistry ; 34(14): 4889-97, 1995 Apr 11.
Article in English | MEDLINE | ID: mdl-7536471

ABSTRACT

The chelating agent diethylenetriaminepentaacetic acid (DTPA) was conjugated site-specifically to the N-terminus of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) by reaction of the protein with DTPA dianhydride at an initial pH of 6.0. The reaction was efficient in that 84% of the starting rhG-CSF was N-terminally modified and could be purified to homogeneity by cation-exchange chromatography. Chelation of 111In by the DTPA-rhG-CSF conjugate was demonstrated by cation-exchange HPLC and thin-layer chromatography. Metal contamination of conjugate preparations, as well as metal-loading onto the conjugate, could be monitored by either cation-exchange HPLC or isoelectric focusing. The 1:1 stoichiometric molar ratio of DTPA to protein for the DTPA-rhG-CSF conjugate was determined by thin-layer chromatography and mass spectrometry, and the localization of the conjugated DTPA moiety was resolved using a peptide mapping procedure. The secondary structure (i.e., alpha-helicity) of the protein was unmodified following conjugation as revealed by circular dichroism. Furthermore, the conjugate induced a similar induction of peripheral WBC counts as unmodified rhG-CSF when injected subcutaneously into hamsters, demonstrating preservation of protein bioactivity. These results reveal a simple and efficient method for conjugating DTPA to protein, via reaction with the dianhydride, to yield a homogeneous and well-defined product. The procedure may prove to be a useful method of labeling growth factors and related proteins while preserving structural and functional integrity.


Subject(s)
Granulocyte Colony-Stimulating Factor/metabolism , Pentetic Acid/metabolism , Animals , Binding Sites , Circular Dichroism , Cricetinae , Granulocyte Colony-Stimulating Factor/chemistry , Humans , Hydrogen-Ion Concentration , Mass Spectrometry , Peptide Mapping , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
8.
Prep Biochem ; 23(3): 375-87, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8395691

ABSTRACT

Covalent structural information on membrane proteins is not easily acquired since it is difficult to obtain pure membrane proteins in sufficient quantities. We have therefore examined the Bio-Rad 491 prep cell continuous elution electrophoresis apparatus as a method for providing the quantities of purified alpha and beta subunits from (Na,K)-ATPase required for these studies. Twenty-four milligrams of crude (Na,K)-ATPase preparation was applied to the prep cell which consisted of a 7% Laemmli separating gel 4.5 cm in length. The prep cell was run under constant power and continuous cooling conditions. Those fractions containing the beta subunit were combined and further purified by wheat germ agglutinin affinity chromatography. Fractions containing the alpha subunit were combined and did not require further purification. The identity and the degree of purity of the proteins obtained using this approach was assessed utilizing SDS-PAGE, amino acid analysis and N-terminal sequencing. This simple and fast method provides approximately 1.8 milligrams of each purified subunit from 24 milligrams of relatively crude microsomes. Recovery of the alpha and beta subunits from the crude (Na,K)-ATPase preparation was estimated to be 28% and 81%, respectively.


Subject(s)
Kidney/enzymology , Microsomes/enzymology , Peptides/isolation & purification , Sodium-Potassium-Exchanging ATPase/chemistry , Amino Acid Sequence , Amino Acids/analysis , Animals , Chromatography, Affinity , Molecular Sequence Data , Sequence Analysis , Sheep
9.
Anal Biochem ; 212(1): 138-42, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8368486

ABSTRACT

The fluorescent cysteine derivative formed upon alkylation of proteins with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) is shown to be unstable under certain conditions. Although previously thought to be an irreversible reaction, the fluorescence and absorbance associated with the ABD-cysteine can be released from the protein by heating at basic pH in the presence of reductants. Reversal of labeling is accelerated by increasing the temperature, pH, and concentration of reductant. Upon reversal of labeling, a free sulfhydryl is regenerated, since the treated protein can subsequently be realkylated (without reductant) to approximately the same level with only ABD-F. The findings reported in this work are relevant to the use of this alkylating reagent prior to any procedure involving heating or long incubation under reducing conditions, including re-electrophoresis or enzymatic deglycosylation. Investigators using ABD-F or structurally similar reagents should be aware that the cysteine derivative formed is not stable under these conditions. The reversal of cysteine derivatization may also be useful as a method for replacing the ABD-cysteine adduct with a label more suitable for subsequent specialized procedures.


Subject(s)
Cysteine/chemistry , Oxadiazoles/chemistry , Alkylation , Animals , Cattle , Dithiothreitol , Drug Stability , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Proteins/chemistry , Serum Albumin, Bovine/chemistry
10.
J Biol Chem ; 268(19): 13914-9, 1993 Jul 05.
Article in English | MEDLINE | ID: mdl-8390982

ABSTRACT

(Na,K)-ATPase is an integral membrane protein responsible for maintaining the Na+ and K+ ion concentration gradients across the plasma membranes of cells. All active (Na,K)-ATPase preparations consist of two subunits, designated alpha and beta. The alpha-subunit is the catalytic subunit and contains the cardiac glycoside binding site. In contrast, the physiological function of the beta-subunit remains unclear although it appears to be involved in the processes of folding, membrane insertion, and stabilization of the alpha-subunit. Previous work has determined the amino acid sequence and disulfide bond arrangements for the beta-subunit from both lamb and dog kidney. In this report, we describe the isolation and structural characterization of the glycan moieties of the beta-subunit from both lamb and dog kidney (Na,K)-ATPase. The three glycosylation sites of these beta-subunits were fractionated using reverse phase chromatography after cleavage of the polypeptide chain with trypsin and thermolysin. Glycopeptides derived from each glycosylation site were analyzed by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry results indicated that the predominant glycoforms at the three glycosylation sites of these beta-subunits were a combination of the tetraantennary glycan form and the unusual glycan form of tetraantennary with a limited number of repeating N-acetyllactosamine units. These results further define the covalent structure for the beta-subunit from both lamb and dog kidney (Na,K)-ATPase and suggest that the beta-subunit may be derived from an adhesion molecule.


Subject(s)
Kidney/enzymology , Membrane Glycoproteins/chemistry , Polysaccharides/chemistry , Sodium-Potassium-Exchanging ATPase/chemistry , Amino Acid Sequence , Animals , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Dogs , Macromolecular Substances , Mass Spectrometry , Membrane Glycoproteins/isolation & purification , Molecular Sequence Data , Polysaccharides/isolation & purification , Sheep , Sodium-Potassium-Exchanging ATPase/isolation & purification , Trypsin
11.
FEBS Lett ; 314(1): 97-100, 1992 Dec 07.
Article in English | MEDLINE | ID: mdl-1333419

ABSTRACT

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.


Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/pharmacology , Sodium-Potassium-Exchanging ATPase/drug effects , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Dogs , Kidney/enzymology , Kinetics , Lysine , Molecular Sequence Data , Peptide Mapping , Potassium Chloride/pharmacology , Protein Conformation , Sodium-Potassium-Exchanging ATPase/metabolism , Structure-Activity Relationship
13.
J Biol Chem ; 267(17): 11777-82, 1992 Jun 15.
Article in English | MEDLINE | ID: mdl-1534802

ABSTRACT

There exists a Mg(2+)-ATPase in transverse tubules which has properties that are very different from other ATPases located in skeletal muscle cells. Several groups have suggested that a 100-kDa glycoprotein is the molecular entity responsible for this Mg(2+)-ATPase activity. In this study we have extended the methods utilized in the purification of this integral membrane glycoprotein. Although the apparent pI (isoelectric point) of this protein is pH 5, most of the net charge is due to the presence of sialic acid on the associated glycan chains. After these residues are removed, the behavior of this protein on an anion exchange column changes dramatically, allowing it to be further purified. Using a combination of the earlier procedures (Kirley, T.L. (1988) J. Biol. Chem. 263, 12682-12689 and Kirley, T. L. (1991) Biochem. J. 278, 375-400.) and the one reported here, purification to specific activities of approximately 400,000 mumol of ATP hydrolyzed/mg of protein/h were obtained and all traces of the 100-kDa protein were removed. The digitonin-solubilized Mg(2+)-ATPase appears to be a dimer of two identical 67-kDa subunits as assessed by high performance size exclusion chromatography. A single diffuse protein band is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at approximately 67 kDa, which reduced to a single tight protein band at 52 kDa after deglycosylation with the following unique N-terminal amino acid sequence: Ala-Lys-Lys-Val-Leu-Pro-Leu-Leu-Leu-Pro- Pro-Leu-Val-X-Ala-Ala-Leu-Gly-Leu-Ala-X-Phe. Therefore, the Mg(2+)-ATPase appears to be an enzyme of very high specific activity, present in small quantities in transverse tubule membranes and unrelated to the known classes of ATPases present in skeletal muscle. The data reported here on the orientation of the transverse-tubule membrane preparations are consistent with the very recent report (Saborido, A., Moro, G., and Megias A. (1991) J. Biol. Chem. 266, 23490-23498) that the Mg(2+)-ATPase is an ecto-enzyme.


Subject(s)
Ca(2+) Mg(2+)-ATPase/chemistry , Glycoproteins/metabolism , Muscles/enzymology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Ca(2+) Mg(2+)-ATPase/metabolism , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Isoelectric Point , Microtubules/metabolism , Molecular Sequence Data , Rabbits
14.
Biochem J ; 283 ( Pt 1): 105-12, 1992 Apr 01.
Article in English | MEDLINE | ID: mdl-1567356

ABSTRACT

Orosomucoid (OMD) contains complex bi-, tri- and tetra-antennary glycan chains. Subfractionation of OMD into three molecular variants using concanavalin A lectin chromatography is based on variations in these complex structures. Standard h.p.l.c. profiles have been developed to analyse the percentage and distribution of the glycoforms present at each glycosylation site in OMD and its molecular variants. The ability to quantify the glycoforms present at each site allows us to extend the earlier results of others and resolve the remaining questions concerning the glycan structures of these variants. Most significantly, the proportions of bi-, tri- and tetra-antennary chains differ at each site for the three molecular variants. The most strongly retained variant from concanavalin A is uniquely capable of possessing biantennary chains at all five sites, whereas the unretained variant is completely devoid of biantennary chains. Only glycosylation site II of the five present is 100% biantennary in the retained and weakly retained variants. In addition, the two gene products of OMD were differentially glycosylated. Molecular masses of the glycoforms were verified by matrix-assisted u.v. laser desorption mass spectrometry. On the basis of the site distribution of oligosaccharides in the variants, efforts were made to understand the factors that control the processing of the carbohydrate chains in OMD. The results indicate that the 'site-directed' model of processing offers the most consistent explanation for the structures seen at the individual glycosylation sites of OMD.


Subject(s)
Orosomucoid/ultrastructure , Amino Acid Sequence , Binding Sites , Chromatography, High Pressure Liquid , Concanavalin A , Glycosylation , Hexoses/isolation & purification , Humans , Molecular Sequence Data , Orosomucoid/metabolism , Peptides/isolation & purification , Polysaccharides , Serine Endopeptidases/metabolism
15.
Biomed Chromatogr ; 6(1): 50-2, 1992.
Article in English | MEDLINE | ID: mdl-1350937

ABSTRACT

Orosomucoid, a member of the lipocalin family, may function in the in vivo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-1-methyl-1,5-benzodiazepine-2,4-dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.


Subject(s)
Anti-Anxiety Agents/isolation & purification , Benzodiazepines , Benzodiazepinones/isolation & purification , Orosomucoid/isolation & purification , Anti-Anxiety Agents/blood , Benzodiazepinones/blood , Chromatography, Affinity , Chromatography, High Pressure Liquid , Clobazam , Female , Humans , Mass Spectrometry
SELECTION OF CITATIONS
SEARCH DETAIL
...