Guillermo Tous - Academia.edu (original) (raw)

Papers by Guillermo Tous

Journal of Neurochemistry - J NEUROCHEM, 1991

Abstract: The bovine γ-aminobutyric acidA/benzodiazepine receptor complex has been purified by a ... more Abstract: The bovine γ-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.

Journal of Liquid Chromatography, 1988

A technique was developed to exchange the backbone -N-H protons in D,O in the native

A method based on amino acid analysis has been developed for monitoring the covalent conjugation ... more A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Journal of Virological Methods, 2007

Adequate biophysical characterization of influenza virions is important for vaccine development. ... more Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID 50 ), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Journal of Liquid Chromatography & Related Technologies, 2008

A new Ultra‐Performance Liquid Chromatography (UPLC) method for assaying paclitaxel and related c... more A new Ultra‐Performance Liquid Chromatography (UPLC) method for assaying paclitaxel and related compounds in plant tissue cultures is described. The method has been shown to be rapid, economical, accurate, linear, precise, and specific, and can be used to assay bulk drug substance (BDS) and determine its chromatographic purity, as well as to assay paclitaxel in process and biomass samples.

Journal of Chromatography A, 1988

A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is ... more A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'4erminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversedphase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

Biotechnology Progress, 2011

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle l... more A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.

Biochemistry, 1998

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been s... more The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Analytical Chemistry, 2007

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biologic... more We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met-and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects. . ‡ Current address: Phyton Biotech Inc., 279 Princeton-Highstown Rd., East Windsor, NJ 08520.

Analytical Biochemistry, 1991

A method based on amino acid analysis has been developed for monitoring the covalent conjugation ... more A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Analytical Biochemistry, 1987

A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus... more A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus consists of a glass vacuum desiccator, a ceramic plate, and a Teflon ring. The method was shown to give reproducible compositions for hydrolysis of human serum albumin and microanalysis of alpha-melanocyte stimulating hormone including the quantitation of as little as one residue of tryptophan. It minimizes sample handling and allows for the simultaneous hydrolysis of a large number of samples.

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoc... more A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.

A technique was developed to exchange the backbone -N-H protons in D,O in the native

A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phe... more A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phenylalanine were synthesized as potential serine protease inhibitors. N-Acetyl derivatives showed irreversible inactivation vis-a-vis subtilisin, while the N-benzoyl ones were specific toward chymotrypsin. The most potent inactivation of chymotrypsin was achieved by a O'-(3,4-epoxybutyl)-L-tyrosine derivative. The inactivation was shown to be stereospecific since a D derivative led to no irreversible inactivation. Placement of the epoxyalkyl group at the carboxy terminus led to potent rapid inactivation. Under these conditions some of the activity was later recovered. The two classes of inactivators (O'-epoxyalkyl and carboxy-epoxyalkyl) appear to operate by different mechanisms. Most importantly, it was found that irreversible inactivation by O'-(epoxyalkyl)-L-tyrosine only resulted if the carboxy terminus was a substrate (i.e. a compound with free carboxy terminus did not lead to inactivation). The ultimate activity kinetic assay (Daniels, S. B.; et al. J. Biol. Chem. 1983, 258, 15046-15053.) indicated that the epoxyalkyl group on the phenolic oxygen had an optimal length of four carbons with respect to the turnover ratio (the ratio of molecules undergoing turnover compared to those that inactivate the enzyme) for chymotrypsin. A different kinetic assay (Ashani, Y.; Wins, P.; Wilson, I. B. Biochim. Biophys. Acta 1972, 284, 427-434.) demonstrated that substratelike turnover was proceeding at considerably slower rates than for the corresponding true substrates and with rate-limiting deacylation of the acyl-enzyme. Amino acid analysis subsequent to acid hydrolysis demonstrated that Met had been selectively alkylated by the O'-(epoxyalkyl)tyrosine derivative. By contrast, alpha-chymotrypsin inactivated with N-benzoyl-L-Phe-2,3-epoxypropyl ester then subjected to amino acid analysis showed no change in the content of any amino acid that would serve as a potential nucleophile to the inhibitor. Yet, the L-Phe content increased, indicating that a covalent bond had been formed between the inhibitor and the enzyme. Either the bond between the inhibitor and the enzyme did not withstand the hydrolytic conditions and/or there was less than 10% decrease in the amino acids with nucleophilic side chains upon inactivation. Finally, two tripeptides containing O'-(epoxyalkyl)-L-tryosines were synthesized [N-(tert-butoxycarbonyl)-L-alanyl-L-alanyl-O'-(2,3-epoxypropyl)-L-tyrosi ne ethyl ester and N-(trifluoroacetyl)-L-valyl-O'-(2,3-epoxypropyl)-L-tyrosyl-L-valine methyl ester] as potential elastase inhibitors and were found to reversibly and competitively inhibit porcine pancreatic elastase.

Journal of Neurochemistry - J NEUROCHEM, 1991

Abstract: The bovine γ-aminobutyric acidA/benzodiazepine receptor complex has been purified by a ... more Abstract: The bovine γ-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.

Journal of Liquid Chromatography, 1988

A technique was developed to exchange the backbone -N-H protons in D,O in the native

A method based on amino acid analysis has been developed for monitoring the covalent conjugation ... more A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Journal of Virological Methods, 2007

Adequate biophysical characterization of influenza virions is important for vaccine development. ... more Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID 50 ), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Journal of Liquid Chromatography & Related Technologies, 2008

A new Ultra‐Performance Liquid Chromatography (UPLC) method for assaying paclitaxel and related c... more A new Ultra‐Performance Liquid Chromatography (UPLC) method for assaying paclitaxel and related compounds in plant tissue cultures is described. The method has been shown to be rapid, economical, accurate, linear, precise, and specific, and can be used to assay bulk drug substance (BDS) and determine its chromatographic purity, as well as to assay paclitaxel in process and biomass samples.

Journal of Chromatography A, 1988

A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is ... more A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'4erminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversedphase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

Biotechnology Progress, 2011

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle l... more A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.

Biochemistry, 1998

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been s... more The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Analytical Chemistry, 2007

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biologic... more We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met-and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects. . ‡ Current address: Phyton Biotech Inc., 279 Princeton-Highstown Rd., East Windsor, NJ 08520.

Analytical Biochemistry, 1991

A method based on amino acid analysis has been developed for monitoring the covalent conjugation ... more A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Analytical Biochemistry, 1987

A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus... more A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus consists of a glass vacuum desiccator, a ceramic plate, and a Teflon ring. The method was shown to give reproducible compositions for hydrolysis of human serum albumin and microanalysis of alpha-melanocyte stimulating hormone including the quantitation of as little as one residue of tryptophan. It minimizes sample handling and allows for the simultaneous hydrolysis of a large number of samples.

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoc... more A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.

A technique was developed to exchange the backbone -N-H protons in D,O in the native

A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phe... more A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phenylalanine were synthesized as potential serine protease inhibitors. N-Acetyl derivatives showed irreversible inactivation vis-a-vis subtilisin, while the N-benzoyl ones were specific toward chymotrypsin. The most potent inactivation of chymotrypsin was achieved by a O'-(3,4-epoxybutyl)-L-tyrosine derivative. The inactivation was shown to be stereospecific since a D derivative led to no irreversible inactivation. Placement of the epoxyalkyl group at the carboxy terminus led to potent rapid inactivation. Under these conditions some of the activity was later recovered. The two classes of inactivators (O'-epoxyalkyl and carboxy-epoxyalkyl) appear to operate by different mechanisms. Most importantly, it was found that irreversible inactivation by O'-(epoxyalkyl)-L-tyrosine only resulted if the carboxy terminus was a substrate (i.e. a compound with free carboxy terminus did not lead to inactivation). The ultimate activity kinetic assay (Daniels, S. B.; et al. J. Biol. Chem. 1983, 258, 15046-15053.) indicated that the epoxyalkyl group on the phenolic oxygen had an optimal length of four carbons with respect to the turnover ratio (the ratio of molecules undergoing turnover compared to those that inactivate the enzyme) for chymotrypsin. A different kinetic assay (Ashani, Y.; Wins, P.; Wilson, I. B. Biochim. Biophys. Acta 1972, 284, 427-434.) demonstrated that substratelike turnover was proceeding at considerably slower rates than for the corresponding true substrates and with rate-limiting deacylation of the acyl-enzyme. Amino acid analysis subsequent to acid hydrolysis demonstrated that Met had been selectively alkylated by the O'-(epoxyalkyl)tyrosine derivative. By contrast, alpha-chymotrypsin inactivated with N-benzoyl-L-Phe-2,3-epoxypropyl ester then subjected to amino acid analysis showed no change in the content of any amino acid that would serve as a potential nucleophile to the inhibitor. Yet, the L-Phe content increased, indicating that a covalent bond had been formed between the inhibitor and the enzyme. Either the bond between the inhibitor and the enzyme did not withstand the hydrolytic conditions and/or there was less than 10% decrease in the amino acids with nucleophilic side chains upon inactivation. Finally, two tripeptides containing O'-(epoxyalkyl)-L-tryosines were synthesized [N-(tert-butoxycarbonyl)-L-alanyl-L-alanyl-O'-(2,3-epoxypropyl)-L-tyrosi ne ethyl ester and N-(trifluoroacetyl)-L-valyl-O'-(2,3-epoxypropyl)-L-tyrosyl-L-valine methyl ester] as potential elastase inhibitors and were found to reversibly and competitively inhibit porcine pancreatic elastase.