DEAMIDATED ACTIVE INTERMEDIATES IN THE IRREVERSIBLE ACID DENATURATION OF RIBONUCLEASE-A

A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH < 1) aqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromatographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa₁. The conversion of RNase-A to RNase-Aa₁ was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa₁ was found to have very nearly the same activity and physico-chemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.     

ON THE REACTIVITY OF CARBOXYL GROUPS OF RIBONUCLEASE-A IN ANHYDROUS METHANOL

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsequently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.     

Regeneration of native structure and biological activity by air oxidation of the reduced bovine trypsin inhibitor (Kunitz)*

Derivatives of the tetraguanidinated bovine trypsin-kallikrein inhibitor lacking up to four amino-acid residues have been prepared by Edman degradation of the amino-terminal Arg-Pro-Asp-Phe sequence. Comparative studies on the native inhibitor, tetraguanidinated inhibitor, and truncated sequences demonstrated that none of the four N-terminal amino-acid residues are essential for the regeneration of native structure and biological activity by air oxidation of the reduced molecule. However residues 2, 3, and 4, although to a different extent, are important for the protein stability and significantly affect the conformation of the tetraguanidinated inhibitor and the rate of the reactivation process. Removal of the terminal positive charge from Arg 1 by diazotization gave a fully active deaminated, tetraguanidinated inhibitor but the lack of the salt bridge interaction between the amino and the carboxyl terminus of the protein prevented the correct folding of the reduced molecule by air oxidation as shown by activity measurements and conformational studies. The stability constants and the standard free energies of binding of the complexes between trypsin and the different inhibitor derivatives have been determined.     

维甲类化合物的研究 IV.双叔丁基苯类化合物的设计合成和构效关系

以３，５双叔丁基４羟基苯基为结构单元设计合成了取代的酰芳胺、芳酯和查尔酮类化合物，模拟全反式维甲酸的分子形状、长度和功能基的空间配置，并研究对细胞诱导分化的活性。结果表明分子一端的疏水性基团，另一端羧基的存在以及分子的共轭性，是呈现诱导分化活性的必要和充分的因素，缺少其中任何一个则失去活性，而且分子的构象、形状和基团的配置也起关键作用。芳酰胺类的Ｎ甲基化产物的稳定构象是两个芳环呈顺式排布，使分子成弯曲状，而不呈反式的伸展形式，以致与全反式维甲酸分子的伸展形状不同，这是它失去活性的原因。本工作发现化合物Ｎｏ４ｆ，４ｇ，７，１３，３２，３７和３８等具有显著的细胞诱导分化作用。     

Role of amino and carboxyl groups of cobrotoxin in the conformational stability and the interaction with acetylcholine receptor

To study the functional involvements of the common interaction of the Leu-1 α-amino group and Asp-58 in cobrotoxin, the lysine ε-amino groups of cobrotoxin were initially guanidinated with o-methylisourea. The α-amino group of Leu-I was then modified with TNBS after the guanidination of cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the β-sheet secondary structure of cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 α-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of cobrotoxin, and the free carboxyl groups in cobrotoxin are conformation-essential. © Munksgaard 1995.     

Structure-function studies on human growth hormone

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmuno-assays using ¹²⁵I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, ¹²⁵I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, ¹²⁵I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radio-receptor assays. One of these, missing the hexapeptide corresponding to residues 135–140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically acitve, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.     

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

EXTENSIVE MODIFICATION OF PROTEIN AMINO GROUPS BY REDUCTIVE ADDITION OF DIFFERENT SIZED SUBSTITUENTS

The amino groups of ovomucoid, lysozyme and ovotransferrin have been extensively alkylated by reacting the proteins with various carbonyl reagents in the presence of sodium borohydride. The extent of modification ranged from 40 to 100%. Essentially monosubstitution was obtained with acetone, cyclopentanone, cyclohexanone and benzaldehyde, while 20–50% disubstitution was obtained with N-butanal and nearly 100% disubstitution was obtained with formaldehyde. Both the methylated and isopropylated derivatives of all three proteins were soluble and retained almost full biochemical activities, but introduction of the larger substituents caused precipitation with lysozyme and ovotransferrin.     

The functional role of histidine and sulfhydryl groups in rat liver thiamine pyrophosphokinase

The functional role of histidine and sulfhydryl groups of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, E.C.2.7.6.2) has been studied by the methods of photooxidation and chemical modification by diethylpyrocarbonate (DEPC) and Ellman's reagent, 5,5' - dithiobis (2 - nitrobenzoic acid) (DTNB). Histidine amino acid residues have been shown to be destroyed during photoinactivation. Titration of the protein with DEPC has established that modification of two histidine groups decreases the catalytic activity of thiamine pyrophosphokinase. Interaction of the subsequent three groups with the reagent does not affect residual activity. Substrates protect thiamine pyrophosphokinase from inactivation. Two -SH groups have been identified in a molecule of thiamine pyrophosphokinase with Ellman's reagent. Modification of only one of them results in complete loss of the enzymatic activity. Treatment of the enzyme with 8M urea has shown no differences in the amount of thiol groups of the native and denatured enzymes. Mg2+ + ATP and somewhat more weakly Mg2+ partially protect thiamine pyrophosphokinase from inhibition by DTNB. In their presence a high degree of enzyme reactivation is observed. Both histidine and sulfhydryl groups are suggested to play an important role in the catalytic mechanism of thiamine pyrophosphokinase.     

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A₂ with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity ($\text{1}\text{4}$1 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Caboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12 – 17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17 – 33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Gluresidues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A₂ are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Influence of some formulation parameters on lysozyme adsorption and on its stability in solution

According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.     

Modification of carboxyl groups in botulinum neurotoxin types A and E

Effects of chemical modification of carboxyl groups of botulinum neurotoxin serotypes A and E were studied by using a water soluble carbodiimide-nucleophile reaction that is highly specific for modifying carboxyl groups of proteins. In both types A and E, increasing levels of the reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and norleucine methyl ester or glycine methyl ester, at pH 4.8 caused increased loss of toxicity. More glycine could be incorporated than norleucine. Amino acid analysis did not reveal modification of any amino acid residue other than carboxyl groups (possible reaction of sulfhydryl groups was not studied). Loss of one carboxyl group did not severely affect toxicity, but modification of three carboxyl groups caused greater than 95% detoxification in both types. Complete detoxification could not be achieved with any amount of the reagents. Modification of three to five carboxyl groups did not affect serological activity.     

Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The major phospholipase A₂ was isolated from Naja naja atra venom by successive chromatography on SP-Sephadex C-25, DEAE-Sephacel, CM-Sephadex C-25 and SP-Sephadex C-25 columns. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 5.2. The specific activity was 3400 U/mg protein, and the ld₅₀ 8 mg/kg mouse. The purified phospholipase A₂ was subjected to lysine modification with cyanate at pH 8.0. After suitable periods (3 and 16 hr), the carbamylated derivatives were separated on a column of DEAE-Sephacel and eight fractions were obtained (DE-1 to DE-8). The results of amino acid analysis showed that one to five Lys-residues were modified. Associated with modification of increasing numbers of Lys-residues were progressive decreases in pI values and marked decreases (3 to > 30-fold) in ld₅₀ values. However, the decrease in enzymatic activity was slight and antigenic specificity was unaffected. The results show a clear dissociation between enzymatic activity and lethal toxicity. The enzyme was also subjected to chemical modification with p-bromophenacyl bromide. Alkylation of the only His-47 at the active site of the phospholipase A₂ destroys both catalytic activity and lethal toxicity, whereas the antigenicity remained unchanged. Although all the native, Lys-modified and His-modified phospholipases A₂ were perturbed by the presence of Ca²⁺ and the difference spectra of Lys-modified DE-6 was similar to that of native phospholipase A₂, the difference spectra of His-modified enzyme differed greatly from that of the native enzyme. The emission intensity of 8-anilinonaphthalenesulfonate-enzyme complex was altered by increasing concentrations of Ca²⁺, and different results were observed at different pH values of the buffer solution, indicating that Ca²⁺ causes pH-dependent conformational changes. The Scatchard plots showed only one kind of specific interaction between 8-anilinonaphthalenesulfonate and native or Lys-modified enzyme (DE-6), and the dissociation constant of Lys-modified DE-6 was similar to that of the native enzyme. On the other hand, the His-modified enzyme lost the ability to bind 8-anilinonaphthalenesulfonate.     

Quantitative structure-activity relationships in amphotericin B derivatives

The quantitative structure-activity relationships studies of amphotericin B and its 16 semisynthetic derivatives obtained by modification at carboxyl and amino groups have been done. The results of five biological tests were subjected to principal component analysis, a numerical method useful in the investigation of large sets of data. For some compounds, also, interaction with lipidic vesicles was investigated by spectroscopic methods. The results obtained indicate that: (i) The presence of positively charged nitrogen atom (protonable or bearing fixed charge) is indispensable for biological activity and antibiotic-sterol interaction; (ii) The lack of free carboxyl group in the molecule favours the differentiation between cholesterol and ergosterol containing cells.     

Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms

Previously we selectively modified His (48), Arg, Lys, Asp, Glu and Trp residues in the basic phospholipase A2 from Naja nigricollis and the acidic phospholipase A2 from N. n. atra snake venoms. Evidence was obtained for the existence of separate but perhaps overlapping sites responsible, respectively, for their enzymatic and pharmacological properties. We have now modified one or two (Tyr 3, Tyr 62 [63], Tyr 3 + 62 [63]) out of the nine tyrosine residues in these enzymes using p-nitrobenzenesulfonyl fluoride. The derivatives were separated by HPLC, and modified residues determined by amino acid analysis. Enzymatic activity was tested on lecithin--Triton mixed micelles, egg yolk and heart and diaphragm homogenates. The N. nigricollis modified derivatives retained a greater percentage of their enzymatic activities than did the N. n. atra derivatives and also a greater percentage of their activity on natural substrates than on lecithin--Triton mixed micelles. The greatest loss in activity resulted when both tyrosines were modified and the least when tyrosine 3 was modified. Modification of tyrosine 62 of N. nigricollis caused a much greater loss of intraventricular lethal potency than of enzymatic activity, whereas modification of tyrosine 3 of N. n. atra increased lethal potency over six-fold while enzymatic activity decreased about 60%. Examples of dissociation between enzymatic and pharmacological potencies were also noted when hemolytic, anticoagulant and cardiotoxicity on isolated ventricular muscle were measured. The extents of phospholipid hydrolysis were relatively low in brain homogenates, synaptic plasma membranes and heart ventricular muscle. However, they were similar for the native enzymes and all of the tyrosine modified derivatives. These tyrosines do not appear to be part of the enzymatic active site, even though they are thought to be associated with substrate and calcium binding. These results strengthen our earlier conclusion that some pharmacological effects of phospholipase A2 are not due to enzymatic hydrolysis, and that there are separate but perhaps partly overlapping sites for enzymatic and pharmacological activities.     

COMPARATIVE THEORETICAL CONFORMATIONAL ANALYSIS OF THE INSULIN A6-A11 PEPTIDES

The insulins of pig, ox, horse and goat differ only by the cyclic A6-A11 peptide which is thought to be an antigenic determinant of the molecule. The structure of the four peptides is investigated by conformational energy calculations in order to verify whether a common backbone conformation can be found for all four species, the antigenic difference being consequently due only to the difference in the side chains exposed to the solvent, or whether each sequence gives rise to a preferential backbone conformation, which would lead to the conclusion that the antigenic difference is conveyed by a more pronounced difference in the molecular structure. From the results of the study on the isolated peptides, it appears that an energetically favourable backbone conformation common to all four species can be found. This conformation is compared with the structure deduced from the X-ray diffraction data available for pig insulin.     

Isolation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30° for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa₂, Aa_1c, Aa_1b, and Aa_1a in order of their elution. With the exception of RNase Aa₂, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa_1a, Aa_1b, and Aa_1c are monodeamidated derivatives of RNase A; RNase Aa₂ contains, in addition, a small amount of a dideamidated component. RNase Aa₂, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67–74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65–72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

STRUCTURAL STUDIES ON STEM BROMELAIN

Stem bromelain was cleaved with cyanogen bromide, and the products were fractionated with and without prior maleylation and sulfitolysis. The fragments that corresponded to the carboxyl-terminal half of the molecule were isolated and nearly completely sequenced. This portion of the enzyme molecule contained one disulfide linkage. A specific cleavage at the amino peptide bonds of that cystine residue by reduction, modification into S-cyano derivatives and exposure to alkali gave important information of the amino terminal sequence. By combining the present data with the previously known partial sequence of the parent molecule, 101 amino acid residues were aligned down to the carboxyl terminus and compared with those of papain. The sequence homology between carboxyl-terminal halves of these two thiol proteases of plant origin was found to be 34.7%.     

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.     

Synthesis of hypoallergenic derivatives of the major allergen Fag t 1 from tartary buckwheat via sequence restructuring

Fag t 1, a legumin-type protein, is the major allergen in tartary buckwheat. In the current study, three recombinant derivatives of Fag t 1, designated as Fag t 1-rs1, Fag t 1-rs2, and Fag t 1-rs3, were constructed via rational design and genetic engineering. However, because of the loss of their native-like folds, the Fag t 1 derivatives failed to bind IgE, and their allergenic activities were reduced. The recombinant hypoallergenic variants are promising vaccine candidates for specific immunotherapy of buckwheat allergy. The unfolding of the Fag t 1 structure reduced its high resistance to gastrointestinal proteolysis and strongly reduced its IgE reactivity. The derivatives showed a more than 90% reduction in allergenic activity compared with rFag t 1. These results suggest that the structure-dependent stability of 11S seed storage proteins is directly related to digestive stability and allergenic potential. Therefore, the destruction of the native conformation is the appropriate strategy to reduce the allergenicity of the cupin family food allergens.