A. Seetharama Acharya

Implication of the structure and stability of disulfide intermediates of lysozyme on the mechanism of renaturation

SummaryThe conformational properties and stability of reduced hen egg lysozyme and those of the disulfide intermediates species formed during renaturation of reduced lysozyme have been reviewed. This information and that related with RNase A and other proteins are interpreted to outline a possible mechanism of renaturation of the reduced protein.

Application of reductive dihydroxypropylation of amino groups of proteins in primary structural studies: identification of phenylthiohydantoin derivative of ε-dihydroxypropyl-lysine residues by high-performance liquid chromatography

The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyanoborohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, epsilon-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of epsilon-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of epsilon-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-epsilon-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the epsilon-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of epsilon-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of epsilon-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.

Inhibition of deoxyhemoglobin S polymerization by glyceraldehyde.

Glyceraldehyde reacts with hemoglobin S in the intact erythrocyte to reduce the degree of polymerization, thereby inhibiting sickling of the erythrocyte. Only five of the 24 amino groups per alpha beta dimer react with glyceraldehyde; the adducts are present as ketoamine structures, formed by Amadori rearrangement of the initial Schiff base adducts on the protein. The reactive amino groups are the epsilon-amino group of Lys-16 of the alpha-chain, and the alpha-amino group of Val-1 as well as the epsilon-amino groups Lys-82, Lys-59, and Lys-120 of the beta-chain. Hybrid tetramers were prepared with the modification only on Lys-16 of the alpha-chain or on the reactive lysine residues of the beta-chain. The former derivative gels at a much higher hemoglobin concentration (23 g/dl) than either the latter derivative (16 g/dl) or unmodified deoxyhemoglobin S (15 g/dl). Thus, the modification at Lys-16 of the alpha-chain is a major factor in the inhibition of sickling by glyceraldehyde.

Reductive hydroxyethylation of the α-amino groups of amidated hemoglobin S

Val-6(β) of hemoglobin S forms the primary site of intertetrameric interaction in the polymerization of deoxy hemoglobin S. However, a number of other intermolecular interactions contribute significantly to the polymerization process as well as to the stability of the polymerized gel. The strong stabilizing influence of Val-6(β) in the polymerization process is reflected in the fact that although a number of mutations at any one of the intermolecular contact regions (or perturbation of these contact regions by chemical modification) result in some increase in the solubility of deoxy hemoglobin S, none of these mutations and/or chemical modifications completely neutralize the polymerizing influence of Val-6(β), i.e., restores the solubility to that of hemoglobin A. Additivity and/or synergy of the solubilizing influence of two or more chemical modification reactions each of which independently increases the solubility may be considered as a possible strategy to restore the solubility of deoxy hemoglobin S to that of hemoglobin A. In the present study, the cumulative solubilizing influence of amidation of Glu-43(β) and hydroxyethylation of α-amino groups of hemoglobin S has been investigated by preparing hemoglobin S with double modification. Modification of Glu-43(β) by amidation with glycine ethyl ester did not influence the reactivity of the α-amino groups of hemoglobin S toward reductive hydroxyethylation, thus permitting the preparation of hemoglobin S with the two modifications. The reductive hydroxyethylation increased the oxygen affinity of amidated hemoglobin S to nearly the same degree as it does on modification of unmodified hemoglobin. In addition, hemoglobin S with double modification has a Hill coefficient that is the same as that of unmodified hemoglobin S, suggesting that the overall quaternary interaction of hemoglobin S with a double modification is nearly the same as the unmodified protein. However, the reductive hydroxyethylation of the amidated hemoglobin S increased the solubility of the protein further. The solubility of hemoglobin S with a double modification is nearly twice that of the unmodified protein and is close to that of 1:1 mixture of hemoglobin S and hemoglobin F. The results demonstrate the additivity of the solubilizing influence of perturbing the quinary interactions at the intermolecular contact regions of deoxy hemoglobin S.

Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the “apparent hydrophobicity” of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.

Reaction of Glyceraldehyde (Aldotriose) with Proteins is a Prototype of Nonenzymic Glycation: Protein Cross-Linking as a Consequence of in Vitro Nonenzymic Glycation

Nonenzymic glycation, a post-translational protein modification reaction, is simply a reflection of the potential of the ‘aldehydic’ function of aldoses to form a reversible Schiff base adduct with the amino groups of proteins and the subsequent intramolecular rearrangement known as Amadori rearrangement to form a more stable ketoamine adduct1. The modification of hemoglobin (Hb) A by glucose, an aldohexose, to form Hb A1c is a continous chemical proccess that occurs in vivo, and is the first known example of nonenzymic glycation2. Besides Hb, many other proteins have been shown to undergo nonenzymic glycation in vivo. One of the long-range chemical consequences of in vivo nonenzymic glycation is the covalent cross-linking of proteins3.

Reversible Dihydroxypropylation of Amino Groups in Proteins: Application in Primary Structural Studies of Streptococcal M-Proteins

M-protein of group A streptococcus is an immunologically diverse antiphagocytic determinant of the bacteria1. In order to better understand the structure-function relationships of the M-proteins, we undertook the determination of their primary structure. The Pep M5 protein, a biologically active, pepsin-derived N-terminal half of the type 5 M protein, was selected for the initial study2. Pep M5 protein contains 6 arginines, 35 lysines, and 47 glutamates, with no methionines and tryptophans, thus limiting the choice for obtaining large peptides for sequence studies to cleavage at its arginyl peptide bonds2. Though arginine specific clostripain appeared to be satisfactory initially, detailed studies of the peptides generated by clostripain digestion of Pep M5 protein revealed that in addition to the major arginine cleavages, digestion occurred at some of the lysine residues. Furthermore, clostripain failed to cleave some of the arginyl bonds quantitatively3. Thus, a better choice for obtaining arginyl peptides appeared to be to take advantage of the high specificity of tryptic cleavage after chemically modifying the e-amino groups of the Pep M5 protein4. The relatively high lysine content of M-protein makes it essential that the reagent used for the modification be very selective to the amino functions and also be capable of derivatizing the lysine residues of the protein completely.

On the renaturation of reduced hen egg white lysozyme containing two blocked sulfhydryl groups.

Formation of native lysozyme from the reduced form involves many pathways in two processes: incorrect pairing of half-cystine residues by oxidation and rearrangement of disulfide (SS) bonds. The energy barrier against suflhydryl (SH)-disulfide interchange of the native or nativelike species thus formed causes accumulation of these species. For example, the enzymatically active isomers containing three presumably native SS bonds and one open SS bond may be thermodynamically favorable over the nonnative isomers and can be formed from reduced lysozyme or lysozyme containing scrambled SS bonds by nonobligatory and flexible pathways. As an extension of these observations formation of nativelike species from reduced lysozyme containing the average of two carboxymethyl (CM)-cysteine was investigated.