ChemInform Abstract Die Volumenänderungen bei der Reaktion von Adenosin (A) und von Adenosin-5'-p... more ChemInform Abstract Die Volumenänderungen bei der Reaktion von Adenosin (A) und von Adenosin-5'-phosphat (AMP) mit NaOH werden gemessen. Die Reaktion von A und AMP dienen als Modelle für eine dilatometrische Untersuchung von Polyriboadenylsäure bei der Protonierung (mit HCl), wobei diese von einer ungeordneten Knäuel-Strukturüber eine l-strängige in eine doppelsträngige Helixübergeht. Dabei tritt eine beträchtliche Vergrösserung des Volumens auf, deren Interpretation versucht wird.
Aggregation of the oxidized heme undecapeptide of mammalian cytochromec in aqueous solution has b... more Aggregation of the oxidized heme undecapeptide of mammalian cytochromec in aqueous solution has been demonstrated by other investigators. Our results indicate that it is the α-amino group on the terminal valine which participates in the aggregation reaction. Fully deaminated (valine and lysine) heme undecapeptide is deaggregated, i.e., it is in the monomeric state, while partially valine-deaminated heme undecapeptide has the circular dichroic spectrum of a mixture of the deaggregated and aggregated species.
Protein 315 from the mouse plasmacytoma, MOPC-315, has been shown by Haimovich et al. (1970) to b... more Protein 315 from the mouse plasmacytoma, MOPC-315, has been shown by Haimovich et al. (1970) to be affinity labeled by :~,N-bromoacetyl-<-N-dinitrophenyl-L-lysine (BADL). Lysine 52 of the heavy chain was shown to be the residue involved in covalent bond formation with this label. On the basis of this reaction, Haimovich et al. (1972) designated the peptide containing this residue as a "site peptide" and assigned this lysine to the antigen-binding region of the protein. In this communication we report studies on the chemical modification of lysyl residues of protein 315 with maleic anhydride in the presence and absence of the protecting hapten, e,N-dinitrophenylaminocaproate, which lead us to conclude that there does not appear to be a protectable lysine in the combining site of this protein. Thus maleylated preparations of protein 315, having essentially every lysyl residue modified, still contain combining sites able to bind hapten with a binding constant identical to that of the unmodified protein. There is essentially no difference between the number of sites remaining in preparations maleylated either in the presence or absence of protecting hapten. In addition, preparations of MOPC-315 protein, maleylated either protected or unprotected, while capable of binding hapten, do not readily incorporate the affinity label BADL, both in terms of rate and extent of incorporation, indicating that lysine 52 of the heavy chain was indeed maleylated. These data indicate that lysine 52, which is involved in affinity labeling with BADL, must be peripheral to the site, since its maleylation does not affect the ability of the site to bind ligand. Further, it cannot be protected against maleylation by ligand.
The volume changes accompanying the reaction of adenosine and of adenosine 5'-phosphate (AMP) wit... more The volume changes accompanying the reaction of adenosine and of adenosine 5'-phosphate (AMP) with sodium hydroxide were measured. Removal of a proton from the adenine ring (near pH 4) produced a volume change of 26.4 ml/mol with adenosine and 23.5 ml/mol with AMP. The phosphate portion of AMP reacts near pH 6 producing a volume change of-2.85 ml/mol. The volume change for the alternate reaction, that of adding a proton to the adenine ring, was calculated from the data for the base-produced volume change. Values of-5.1 ml/rnol for adenosine and-2.2 ml/mol for AMP were obtained. The reactions of adenosine and AMP were used as models for a dilatometric study of polyriboadenylic acid (poly A). When poly A is allowed to react with hydrochloric acid from pH 9 to 3, at least three processes take place. The first is the protonation of adenine. The second is a transition from a random coil to a helical single strand. The third is the formation of the doublestranded helical complex. The second and third processes may occur simultaneously. The measured volume change was shown to be largely the sum of the second and third processes and is as high as 44.1 mljmol of acid reacted. A probable explanation for the large volume increase is the loss of water of hydration accompanying the transition to the helical state. he study of volume changes associated with the (I) This work was supported by a Biomedical Sciences Support Grant (2) I<.
Determinations were made of the amino acid residues involved as contact residues in the hapten co... more Determinations were made of the amino acid residues involved as contact residues in the hapten combining site of rabbit antibodies raised against p-azophenylphosphorylcholine by reacting antibody with either diazoacetamide or maleic anhydride,, followed by glyoxal reagent. The effect of the reaction on ability of the antibody to bind hapten was followed. Reactions were carried out both in the absence and in the presence of the hapten p-nitrophenylphosphorylcholine. A loss of binding activity coupled with the demonstration that the presence of hapten during modification could reduce the loss of activity was taken as evidence that the type of amino acid modified was in the binding site. Glyoxalation of about 70~,~, of the total arginine per molecule (under conditions in which l.vsyl residues were blocked by maleylation) reduced the number of active binding sites in all of the antibody preparations tested. As much as a 447.0 loss of sites was observed in one case. These losses could be partially prevented by carrying out the reaction in the presence of bapten. Arginine. therefore. appears to be a part of the combining site of a significant portion of the molecules of each antibody preparation studied. Esterification of about 20°,,0 the carboxyl groups with diazoacetamide resulted in a decrease in binding capacity (-24~,~) which was protectable by hapten. Although extensive modification of carboxyl groups was not achieved, these results point to the probability that a glutamyl or asparyl residue is in the site. Maleylation of nearly atl of the lysine present with maleic anyhdride was virtually ineffective in reducing the binding capacity of these antibodies, thus indicating the absence of lysine in the site. We conclude that a significant portion of the anti-p-azophenylphosphorylcholine antibody population has a positively charged arginyl residue, and a negatively charged carboxylate group, in the site. These residues are those which provide for electrostatic interaction of the site with the negatively charged phosphoryl group and the positively charged quaternary nitrogen of the p-azophenylphosphorylcholine hapten. l.Nr'l'R OD U CT ION
Abstract The extent to which several polypeptidyl proteins (polyvalyl and polyglycyl ribonuclease... more Abstract The extent to which several polypeptidyl proteins (polyvalyl and polyglycyl ribonuclease, polyvalyl, poly-t-leucyl and polyglycyl α-chymotrypsin, and poly-t-leucyl bovine serum albumin) aggregate has been determined in an attempt to show that these protein derivatives are useful as experimental models for studying hydrophobic bonding. Between 0° and room temperature aqueous solutions of the modified proteins are predominately monomeric. However, among those derivatives modified with nonpolar amino acid residues, a highly aggregated fraction amounting to approximately 10% of the total protein was often present. The particle size of this fraction was large enough to produce a visible turbidity. Lowering the temperature reduced the size of the aggregate, thus causing the solution to clear. This temperature dependence of the aggregation is characteristic of material presumed to be undergoing hydrophobic bonding. Details concerning the aggregation reactions were obtained through use of the ultracentrifuge. At higher temperatures (above approximately 30°) the monomeric fractions of all of the nonpolar derivatives polymerize. The polymerization is dependent on temperature and pH in a manner indicating the involvement of hydrophobic forces. In contrast to the behavior of the nonpolar polypeptidyl proteins, the polyglycyl proteins, having as many or more added residues, were totally monomeric at temperatures from 0° to nearly 100°. They were not only more stable to heat than the nonpolar derivatives, but also more stable than the corresponding unmodified proteins. For example, chymotrypsin which normally exists as a dimer or trimer (as high as the hexamer at low ionic strength) becomes monomeric when glycine peptides are attached. Chymotrypsin precipitates out of solution at about 65°, but solutions of polyglycyl chymotrypsin can be brought almost to the boiling point without evidence of aggregation. Based on the behavior of the derivatives of α-chymotrypsin and ribonuclease, it is suggested that adding glycine to other proteins may also yield a monomeric species. Similarly, attaching nonpolar peptides should produce an aggregate. The modified proteins were characterized by amino acid and end group analysis. A comparison of the aggregated and monomeric fractions of the polyvalyl proteins showed that the chymotrypsin derivatives differed with respect to the extent of modification. There was no detectable difference between the two ribonuclease fractions.
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin le... more A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin le... more A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.
ChemInform Abstract Die Volumenänderungen bei der Reaktion von Adenosin (A) und von Adenosin-5'-p... more ChemInform Abstract Die Volumenänderungen bei der Reaktion von Adenosin (A) und von Adenosin-5'-phosphat (AMP) mit NaOH werden gemessen. Die Reaktion von A und AMP dienen als Modelle für eine dilatometrische Untersuchung von Polyriboadenylsäure bei der Protonierung (mit HCl), wobei diese von einer ungeordneten Knäuel-Strukturüber eine l-strängige in eine doppelsträngige Helixübergeht. Dabei tritt eine beträchtliche Vergrösserung des Volumens auf, deren Interpretation versucht wird.
Aggregation of the oxidized heme undecapeptide of mammalian cytochromec in aqueous solution has b... more Aggregation of the oxidized heme undecapeptide of mammalian cytochromec in aqueous solution has been demonstrated by other investigators. Our results indicate that it is the α-amino group on the terminal valine which participates in the aggregation reaction. Fully deaminated (valine and lysine) heme undecapeptide is deaggregated, i.e., it is in the monomeric state, while partially valine-deaminated heme undecapeptide has the circular dichroic spectrum of a mixture of the deaggregated and aggregated species.
Protein 315 from the mouse plasmacytoma, MOPC-315, has been shown by Haimovich et al. (1970) to b... more Protein 315 from the mouse plasmacytoma, MOPC-315, has been shown by Haimovich et al. (1970) to be affinity labeled by :~,N-bromoacetyl-<-N-dinitrophenyl-L-lysine (BADL). Lysine 52 of the heavy chain was shown to be the residue involved in covalent bond formation with this label. On the basis of this reaction, Haimovich et al. (1972) designated the peptide containing this residue as a "site peptide" and assigned this lysine to the antigen-binding region of the protein. In this communication we report studies on the chemical modification of lysyl residues of protein 315 with maleic anhydride in the presence and absence of the protecting hapten, e,N-dinitrophenylaminocaproate, which lead us to conclude that there does not appear to be a protectable lysine in the combining site of this protein. Thus maleylated preparations of protein 315, having essentially every lysyl residue modified, still contain combining sites able to bind hapten with a binding constant identical to that of the unmodified protein. There is essentially no difference between the number of sites remaining in preparations maleylated either in the presence or absence of protecting hapten. In addition, preparations of MOPC-315 protein, maleylated either protected or unprotected, while capable of binding hapten, do not readily incorporate the affinity label BADL, both in terms of rate and extent of incorporation, indicating that lysine 52 of the heavy chain was indeed maleylated. These data indicate that lysine 52, which is involved in affinity labeling with BADL, must be peripheral to the site, since its maleylation does not affect the ability of the site to bind ligand. Further, it cannot be protected against maleylation by ligand.
The volume changes accompanying the reaction of adenosine and of adenosine 5'-phosphate (AMP) wit... more The volume changes accompanying the reaction of adenosine and of adenosine 5'-phosphate (AMP) with sodium hydroxide were measured. Removal of a proton from the adenine ring (near pH 4) produced a volume change of 26.4 ml/mol with adenosine and 23.5 ml/mol with AMP. The phosphate portion of AMP reacts near pH 6 producing a volume change of-2.85 ml/mol. The volume change for the alternate reaction, that of adding a proton to the adenine ring, was calculated from the data for the base-produced volume change. Values of-5.1 ml/rnol for adenosine and-2.2 ml/mol for AMP were obtained. The reactions of adenosine and AMP were used as models for a dilatometric study of polyriboadenylic acid (poly A). When poly A is allowed to react with hydrochloric acid from pH 9 to 3, at least three processes take place. The first is the protonation of adenine. The second is a transition from a random coil to a helical single strand. The third is the formation of the doublestranded helical complex. The second and third processes may occur simultaneously. The measured volume change was shown to be largely the sum of the second and third processes and is as high as 44.1 mljmol of acid reacted. A probable explanation for the large volume increase is the loss of water of hydration accompanying the transition to the helical state. he study of volume changes associated with the (I) This work was supported by a Biomedical Sciences Support Grant (2) I<.
Determinations were made of the amino acid residues involved as contact residues in the hapten co... more Determinations were made of the amino acid residues involved as contact residues in the hapten combining site of rabbit antibodies raised against p-azophenylphosphorylcholine by reacting antibody with either diazoacetamide or maleic anhydride,, followed by glyoxal reagent. The effect of the reaction on ability of the antibody to bind hapten was followed. Reactions were carried out both in the absence and in the presence of the hapten p-nitrophenylphosphorylcholine. A loss of binding activity coupled with the demonstration that the presence of hapten during modification could reduce the loss of activity was taken as evidence that the type of amino acid modified was in the binding site. Glyoxalation of about 70~,~, of the total arginine per molecule (under conditions in which l.vsyl residues were blocked by maleylation) reduced the number of active binding sites in all of the antibody preparations tested. As much as a 447.0 loss of sites was observed in one case. These losses could be partially prevented by carrying out the reaction in the presence of bapten. Arginine. therefore. appears to be a part of the combining site of a significant portion of the molecules of each antibody preparation studied. Esterification of about 20°,,0 the carboxyl groups with diazoacetamide resulted in a decrease in binding capacity (-24~,~) which was protectable by hapten. Although extensive modification of carboxyl groups was not achieved, these results point to the probability that a glutamyl or asparyl residue is in the site. Maleylation of nearly atl of the lysine present with maleic anyhdride was virtually ineffective in reducing the binding capacity of these antibodies, thus indicating the absence of lysine in the site. We conclude that a significant portion of the anti-p-azophenylphosphorylcholine antibody population has a positively charged arginyl residue, and a negatively charged carboxylate group, in the site. These residues are those which provide for electrostatic interaction of the site with the negatively charged phosphoryl group and the positively charged quaternary nitrogen of the p-azophenylphosphorylcholine hapten. l.Nr'l'R OD U CT ION
Abstract The extent to which several polypeptidyl proteins (polyvalyl and polyglycyl ribonuclease... more Abstract The extent to which several polypeptidyl proteins (polyvalyl and polyglycyl ribonuclease, polyvalyl, poly-t-leucyl and polyglycyl α-chymotrypsin, and poly-t-leucyl bovine serum albumin) aggregate has been determined in an attempt to show that these protein derivatives are useful as experimental models for studying hydrophobic bonding. Between 0° and room temperature aqueous solutions of the modified proteins are predominately monomeric. However, among those derivatives modified with nonpolar amino acid residues, a highly aggregated fraction amounting to approximately 10% of the total protein was often present. The particle size of this fraction was large enough to produce a visible turbidity. Lowering the temperature reduced the size of the aggregate, thus causing the solution to clear. This temperature dependence of the aggregation is characteristic of material presumed to be undergoing hydrophobic bonding. Details concerning the aggregation reactions were obtained through use of the ultracentrifuge. At higher temperatures (above approximately 30°) the monomeric fractions of all of the nonpolar derivatives polymerize. The polymerization is dependent on temperature and pH in a manner indicating the involvement of hydrophobic forces. In contrast to the behavior of the nonpolar polypeptidyl proteins, the polyglycyl proteins, having as many or more added residues, were totally monomeric at temperatures from 0° to nearly 100°. They were not only more stable to heat than the nonpolar derivatives, but also more stable than the corresponding unmodified proteins. For example, chymotrypsin which normally exists as a dimer or trimer (as high as the hexamer at low ionic strength) becomes monomeric when glycine peptides are attached. Chymotrypsin precipitates out of solution at about 65°, but solutions of polyglycyl chymotrypsin can be brought almost to the boiling point without evidence of aggregation. Based on the behavior of the derivatives of α-chymotrypsin and ribonuclease, it is suggested that adding glycine to other proteins may also yield a monomeric species. Similarly, attaching nonpolar peptides should produce an aggregate. The modified proteins were characterized by amino acid and end group analysis. A comparison of the aggregated and monomeric fractions of the polyvalyl proteins showed that the chymotrypsin derivatives differed with respect to the extent of modification. There was no detectable difference between the two ribonuclease fractions.
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin le... more A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin le... more A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.
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Papers by Leon Krausz