Studies on the status of free amino and carboxyl groups in cobrotoxin

The status of free amino groups in cobrotoxin was studied by stepwise modification with trinitrobenzene sulfonate. Lys-27 was selectively modified without altering the activity of cobrotoxin. However, complete loss of activity was observed when Lys-27 and Lys-47 were trinitrophenylated, suggesting that the epsilon-amino group of Lys-47 is essential for the activity of cobrotoxin. The alpha-amino group of N-terminal leucine had no correlation with activity, demonstrated by the guanidination of the lysine residues with O-methylisourea followed by trinitrophenylation of the alpha-amino group. The carboxyl groups in cobrotoxin were modified with glycine methyl ester after activation with water-soluble carbodiimide. Six out of seven free carboxyls reacted in the absence of guanidine.HCl without altering the biological activity. When the remaining carboxyl was modified in the presence of 5 M guanidine.HCl, the resulting toxin was devoid of activity. This "buried" carboxyl is essential for activity and was identified as the gamma-carboxyl group of Glu-21.     

Effect of Chemical Modifications on the Gushing-inducing Activity of a Hydrophobic Protein Produced by a Nigrospora sp

A beer-gushing inducing protein (NGF) produced by Nigrospora sp. No. 207 was found to be characterized by a relatively higher content of hydrophobic amino acid residues, a considerably high content (about 10 %) of half-cystine residue, and the lack of tyrosine, tryptophan, methionine and histidine. Cleavage of disulfide bonds in NGF, either oxidative or reductive, resulted in complete loss of the gushing-inducing activity, indicating that the disulfide bonds are essential for exhibiting the activity.

Modification of amino groups in NGF with maleic anhydride or O-methylisourea did not affect the gushing activity, whereas modification with tri nitrobenzene sulfonate reduced the activity to some extent. Modification of arginine-guanidino groups in NGF with phenyl- glyoxal markedly reduced the gushing activity. When the free carboxyl groups of NGF were modified by incorporating glycine or tyrosine, the gushing activity was also drastically destroyed, implying that the acidic groups of the NGF protein are important for the gushing activity.

It appeared that maintenance of a definite molecular conformation is essential for the gushing-inducing activity of this hydrophobic protein.     

Role of basic residues of human lactoferrin in the interaction with B lymphocytes

We have previously demonstrated that lactoferrin was incorporated into B lymphocytes and that a trypsin treatment for a short period reduced the number of lactoferrin molecules incorporated into B lymphocytes. An N-terminal sequence analysis revealed that the mild trypsin treatment had cleaved the three N-terminal amino acids, Gly1-Arg2-Arg3. Chemical conjugation of lost sequence analogue Gly-Arg-Arg-Gly with the mildly digested lactoferrin recovered the interaction with B lymphocytes, while conjugation of acetyl-Arg-Arg-Gly, a deamino analogue of Gly-Arg-Arg-Gly, did not recover the interaction. This shows that the N-terminal basic region containing N-terminal Gly played an important role in the interaction with B lymphocytes. Acylation of the amino groups of lactoferrin also significantly reduced the interaction with B lymphocytes, and an O-methylisourea treatment of the amino groups, which preserved the positive charge, hardly affected the interaction. These results suggest that both the N-terminal basic region and the basic characteristics of the whole molecule contributed to its interaction with B lymphocytes.     

A chemical approach to the fine structure of biomolecular complexes: the amino terminal region of the 50S ribosomal "A" protein from Bacillus stearothermophilus.

An experimental approach and methodology are described for determining the reactive properties and ionization constants of individual functional groups of proteins within biomolecular complexes. The ionization constants and reactivities of the methionyl-l amino terminus and the lysyl-3 residue of the alanine rich 50S ribosomal "A" protein from Bacillus stearothermophilus have been determined by an extension of the competitive labeling technique used by H. Kaplan, K. J. Stevenson, and B. S. Hartley ((1971), Biochem. J. 124, 289-299). This approach employs (1-14C)- and (3H)acetic anhydride in a double-labeling procedure. In 0.1 M KCl-0.02 M Mg2+-0.05 M Veronal at 10 degrees the methionyl-l amino terminus has a pKa of 7.5 and is exposed on the surface of the ribosome. The lysyl-3 has a pKa of 10 and is also exposed to solvent at the surface of the 50S subunit. Based on a linear free energy relationship (Bronsted plot) obtained with a series of standard amines the methionyl amino terminus has a substantially higher reactivity than expected from its ionization constant. The lysyl epsilon-amino group has the expected reactivity. The abnormally high reactivity of the methionyl amino terminus can only be accounted for by a specific interaction with other functional groups in the ribosome. These data support the proposal that the charged state of this residue is important in the structure and function of the "A" protein at the surface of the ribosome.     

Pyrogenic Responses to Staphylococcal Enterotoxins A and B in Cats

Pyrogenic responses, ranging up to 4.8 F, were induced in cats by oral administration of highly purified staphylococcal enterotoxin B in doses from 10 to 100 mug/kg. Fever was a more sensitive indicator of intoxication than was emesis. Highly purified preparations of enterotoxin A, whether administered intravenously (0.01 to 1.0 mug/kg), orally (10 to 25 mug/kg), or into the cerebral ventricles (0.005 to 0.020 mug in 0.20 ml), were also pyrogenic in cats. Tolerance to the pyrogenic activity was produced by repeated intravenous injection of a given dose of enterotoxin A but not by repeated intracerebroventricular injection. Enterotoxin A was more potent than enterotoxin B after intravenous injection in causing both fever and emesis. Cross-tolerance could not be demonstrated between enterotoxin A and enterotoxin B or Salmonella typhosa endotoxin. This lack of cross-tolerance plus the inability of large oral doses (100 to 4,700 mug/kg) of endotoxin to cause fever or emesis indicate that the reported responses were attributable to the specific toxins administered and not to contamination by other pyrogens.     

Role of charged groups in factor XI/XIa activity

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.     

Guanidination of ovine luteinizing hormone and effects on activity.

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.     

Identification of a novel pyridoxal 5'-phosphate binding site in adenosylcobalamin-dependent lysine 5,6-aminomutase from Porphyromonas gingivalis

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversion of D-lysine with 2,5-diaminohexanoate and of L-beta-lysine with 3,5-diaminohexanoate. The coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP). In the proposed chemical mechanism, AdoCbl initiates the formation of substrate radicals, and PLP facilitates the radical rearrangement by forming an external aldimine linkage with the epsilon-amino group of a substrate, either D-lysine or L-beta-lysine. In the resting enzyme, an internal aldimine between PLP and an essential lysine in the active site facilitates productive PLP binding and catalysis. We present here biochemical, biophysical, and site-directed mutagenesis experiments, which document the existence of an essential lysine residue in the active site of 5,6-LAM from Porphyromonas gingivalis. Reduction of 5,6-LAM with NaBH(4) rapidly inactivates the enzyme and shifts the electronic absorption band from 420 to 325 nm. This is characteristic of the reduction of an aldimine linkage between the carbonyl group of PLP and the epsilon-amino group of a lysine residue. The reduced peptide was identified by Q-TOF/MS and further confirmed by Q-TOF/MS/MS sequencing. We show that lysine 144 in the small subunit of 5,6-LAM is the essential lysine residue. Lysine 144(beta) is separated by only 11 amino acids from histidine 133(beta), which forms a part of the "base-off"-AdoCbl binding motif. The sequence of the novel PLP-binding motif is conserved in 5,6-LAM from Clostridium sticklandii and P. gingivalis, and it is distinct from all known PLP-binding motifs. Mutation of lysine 144(beta) to glutamine led to K144Q(beta)-5,6-LAM, which displayed no enzymatic activity and no absorption band corresponding to an internal PLP-aldamine. In summary, we introduce a novel PLP-binding motif, the first to be discovered in an AdoCbl-dependent enzyme.     

Unusual chemical properties of the amino groups of insulin: implications for structure-function relationship.

The chemical properties of the three amino groups of insulin were obtained at 10 and 37 degrees C using the competitive labelling technique with acetic anhydride as the labelling reagent. At 10 degrees C, pK values of 7.9, 7.2, and 7.8 were found for the glycyl A1, phenylalanyl B1, and lysyl B29 amino groups. When compared with standard amino compounds by means of a Br?nsted plot, the two amino-termini were found to be 'super-reactive' and the lysyl epsilon-amino group buried. In the presence of carbon dioxide at physiological pH values, all three amino groups became much less reactive indicating that they had reacted to form carbamino derivatives. Above pH 8 the reactivities of the glycyl amino terminus and epsilon-amino group increase sharply indicating that insulin is undergoing a conformational change which is most likely a change in its association state. At 37 degrees C the amino groups do not titrate normally but exhibit sharp increases in reactivity over the physiological pH range with the midpoints in the pH reactivity profiles between pH values of 7.0 and 7.3. This behaviour is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. It is concluded that at physiological pH and temperature all three amino groups are deprotonated.     

Structure-function relationships of sea anemone toxin II from Anemonia sulcata

Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in its toxic action on crabs and mice and in its binding properties to the Na+ channel of rat brain synaptosomes. Guanidination of th epsilon-amino groups of lysines 35, 36, and 46 with O-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties. Either acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three epsilon-amino groups and of the alpha-amino function of Gly produced an almost complete loss of toxicity and a considerable decrease in the binding activity. Iodination of the toxin on His induced practically no loss of toxic or binding properties. Carbethoxylation of both histidines 32 and 37 with diethyl pyrocarbonate provoked an important decrease of both the toxicity and the binding activity. Modifications of the guanidine side chain of Arg with 1,2-cyclohexanedione fully destroyed both toxicity and binding of the toxin to the Na+ channel. Modification of the carboxylate functions of Asp, Asp, and of the COOH-terminal Gln with glycine ethyl ester in the presence of a soluble carbodiimide completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged. The antagonist character of this carboxylate-modified derivative was further confirmed by electrophysiological and Na+ flux experiments. The theoretical and practical significance of these results are discussed.     

Site-specific biotinylation. A novel approach and its application to endothelin-1 analogs and PTH-analog.

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the epsilon-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the epsilon-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET-1 analog containing epsilon-biotinylaminocaproyllysine at position-9. This peptide, with Kd = 0.08 nM, has 1000-fold higher affinity for the ETA receptor than the commercially available N alpha-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.     

Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy

Secondary and tertiary structural parameters of two functionally and serologically related proteins, staphylococcal enterotoxins B and C1, have been determined by using circular dichroism and fluorescence spectroscopy. The secondary structures derived from the respective far-UV circular dichroic spectra were 9.5% alpha-helix, 55.0% beta-pleated sheets, 16.5% beta-turns, and 19.0% random coils for enterotoxin B and 15.0% alpha-helix, 38.0% beta-pleated sheets, 25.5% beta-turns, and 21.5% random coils for staphylococcal enterotoxin C1. The values matched well with the secondary structures derived from the amino acid sequences (Chou and Fasman method). Seven antigenic sites have been predicted for both staphylococcal enterotoxins B and C1 by using the hydrophilicity and the secondary structure information. Three of these antigenic sites appear similar. Fluorescence quantum yield of the single tryptophan residue (Trp-197) of both the enterotoxins showed the tryptophan residue in staphylococcal enterotoxin B to be approximately 46% more fluorescent than in staphylococcal enterotoxin C1. Tryptophan fluorescence quenching by the surface quencher I- and the neutral quencher acrylamide revealed that the single tryptophan residue in each of the enterotoxins is buried in the protein matrix and is not accessible to the surface quencher I-. The tryptophan residue in staphylococcal enterotoxin C1 is 14% less accessible to acrylamide than in staphylococcal enterotoxin B. The data, in general, reflect several similarities and significant differences between the two related enterotoxins.     

Analysis of receptor-binding site in Escherichia coli enterotoxin

Heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and cholera enterotoxin are both composed of A and B subunits. The A subunit is an enzymatically active ADP-ribosylating subunit, while the B subunit, consisting of 103 amino acids, binds the toxin to a receptor, GM1-ganglioside, on the cell surface. A mutant isolated after treatment of E. coli producing heat-labile enterotoxin with N-methyl-N'-nitro-N-nitrosoguanidine produces a B subunit that is unable to bind to ganglioside. This subunit was purified and its primary amino acid sequence was determined. It differed from the native B subunit in only one amino acid at position 33; namely it had aspartate instead of glycine at position 33 from the N terminus. Thus glycine at position 33 from the N terminus of the B subunit is important for binding the B subunit to the ganglioside receptor.     

1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation

Reductive alkylation of lysyl epsilon-amino groups with sugars (1-deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58-67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1-deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity.     

Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement

Lysine epsilon-amino groups of human factor H were selectively converted to guanidino groups by treatment with 0.1 M O-methylisourea at pH 10.4. Guanidination resulted in a dose-dependent decrease in the capacity of the regulatory protein to accelerate decay dissociation of P-stabilized amplification C3 convertase sites, to serve as a co-factor for cleavage of cell-bound C3b by I, and to compete for binding of 125I-untreated H to C3b. Modification of approximately 75% lysine epsilon-amino groups suppressed 97% of H functional activity. Biochemical analysis of native H demonstrated a total carbohydrate content of 18.5% (w/w) and the presence in the molecule of 11 biantennary oligosaccharidic chains of the N-acetyl-lactosaminic type. Total desialation of H by using Clostridium perfringens neuraminidase, and total deglycosylation of desialated H by using beta-endo-N-acetylglucosaminidase resulted in a 1.5- to 2-fold increase in H activity on a weight basis. Deglycosylation did not alter the capacity of H to discriminate between activating and nonactivating surfaces of the alternative pathway. Thus, lysine residues are important determinants of the binding capacity of H for cell-bound C3b, whereas the carbohydrate portion of the molecule is not required for the regulatory function of the protein on the amplification C3 convertase.     

The complete amino acid sequence of staphylococcal enterotoxin C1

This report presents the complete amino acid sequence of staphylococcal enterotoxin C1. It has a total of 239 amino acids and a calculated Mr = 27,500. Reaction of the native toxin with trypsin produced five peptides, designated A through E. Their structures were determined after further fragmentation with cyanogen bromide, trypsin, and chymotrypsin. Overlap peptides were prepared by cleavage at the two half-cystine residues, and by the reaction of enterotoxin C1 with hydroxylamine. The latter procedure was employed when it was found that one of the three asparaginyl-glycine loci in the toxin is refractory to direct sequencing. The sequence is compared to staphylococcal enterotoxin B. Extensive homology is found, particularly in the carboxyl-terminal region. However, the segment spanned by the disulfide bond in enterotoxin C1 is three residues shorter than the corresponding segment in the B variant.     

Interaction of pyridoxal-5-phosphate with human serum albumin and pancreatic ribonuclease

Pyridoxal-5-phosphate (in a lesser degree, pyridoxal) interacts with both non-protonated and protonated exposed epsilon-amino groups of lysine residues and with alpha-amino groups in human serum albumin and pancreatic ribonuclease A. The reaction of Schiff base formation proceeds within a wide pH range--from 3.0 to 12.0. At a great pyridoxal-5-phosphate excess in ribonuclease A in neutral or slightly acidic aqueous media all the ten epsilon-amino groups of lysine residues and the alpha-amino groups of Lys-1 become modified. The formation of aldimine bonds of pyridoxal-5-phosphate with protonated amino groups in acidic media is determined by ionization of its phenol hydroxyl and phosphate residues. Acetaldehyde, propionic aldehyde and pyridine aldehyde interact only with non-protonated amino groups of the proteins. The equilibrium constants of pyridoxal-5-phosphate and other aldehydes binding to proteins and amino acids were determined. The rate constants of Schiff base formation for pyridoxal-5-phosphates with some amino acids and primary sites of proteins for direct and reverse reactions were calculated.     

Synthesis and asymmetric reducing performance of chitin/dihydronicotinamide conjugates having glycine or L-leucine spacer arms

Chitin/dihydronicotinamide conjugates having glycine or L-leucine spacer arms have been prepared and evaluated as asymmetric reducing agents. N-Nicotinoylglycine and N-nicotinoyl-L-leucine were synthesized and coupled with the amino group of water-soluble 50%-deacetylated chitin. The remaining free amino groups were acetylated, and the nicotinamide groups were transformed into dihydronicotinamide moieties by quaternization followed by reduction. The resulting L-leucine-containing conjugate reduced ethyl benzoylformate efficiently with high chemical yield and asymmetric selectivity, whereas the glycine-containing conjugate gave only poor results. The recovered L-leucine-containing conjugate was reduced to regenerate the dihydronicotinamide structure and could be used again. The L-leucine residue has thus proved suitable as a spacer arm to achieve a high reducing performance.     

Trinitrophenylation of bovine growth hormone.

Bovine growth hormone was chemically modified with picryl sulfonic acid, at pH 8.4 during 2 and 5 min of reaction. The N-terminal residue provides the most reactive amino group followed by the epsilon-amino groups of lysine 179 and lysines 143, 69, 111, 170 and 166 in decreasing order. These results agree with those obtained previously with equine growth hormone, except that residue 156 is not modified in bovine growth hormone. An important decrease in biological activity occurs between 2 and 5 min of reaction without sensible modification in the alpha-helix content of the molecule.     

Structure-activity relationships of vasoactive peptides derived from fibrin or fibrinogen degraded by plasmin

The amino acid sequences of three vasoactive peptides which we previously isolated from fibrin(ogen) degraded by plasmin (EC 3.4.21.7) have been determined. Each peptide originates from a different chain of the fibrinogen molecule. Comparison with other studies shows that the two peptides having the permeability-increasing effect arise from regions of the fibrinogen' molecule that are easily accessible to plasmin attack. The third peptide, which has a slight vasoconstrictor activity, is part of fragment E released from the fibrinogen molecule after plasmin degradation. The two peptides having permeability-increasing effect lose their carboxyl-terminal lysine after interaction with carboxypeptidase B (EC 3.4.12.3) with a concomitant loss of activity. One of these peptides (6A) is resistant to tryptic digestion, while the second peptide (6D) is easily split into two inactive fragments. Complete deamination or modification of the free amino groups (carbamylation, methylation) almost completely abolishes their activity, whereas selective modification of only the free ϵ-amino groups of lysine does not. Modification of the Trp residue in one of these peptides (6D) increased its activity. These findings show that a carboxylterminal lysine with a free ϵ-amino group as well as an unblocked N-terminal residue are essential for their activity. Proline, which is situated near or at the middle of both peptides, is also apparently essential indicating that a specific conformation might be required for physiological activity.