Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Studies on the osmotic resistance and the viability of amidinated erythrocytes]

The present studies are concerned with properties of amidinated erythrocytes. The reactions of dimethyladipimidate with proteins in solution and red blood cells, respectively, result in an intermolecular cross-linking. Following an amidination of human serum albumin or human gamma-globulin cross-linked products of increased molecular weight have been demonstrated by polyacrylamide gel and immune electrophoresis. Human erythrocytes previously amidinated intensely, exhibit a restricted motility of membrane particles and cross-linked hemoglobin. Intensely amidinated erythrocytes are resistant against distilled water, and they do no longer agglutinate. The findings presumably indicate an increased permeability of the amidinated red cell membrane. The glycolytic activity was found to be normal in moderately amidinated erythrocytes. In comparison with normal red blood cells, previously moderately amidinated erythrocytes of the rat become sequestered more quickly after re-injection into the vascular system.     

Reassembly of a regularly arranged protein in the cell wall of Lactobacillus buchneri and its reattachment to cell walls: chemical modification studies

Regularly arranged protein (RA protein) isolated from the cell wall of Lactobacillus buchneri was chemically modified by amidination, acetylation, succinylation, and amidation. The modified RA proteins were examined for their ability to reassemble into a regular array and to reattach to the cell walls from which the regular array had been detached. Only amidinated RA protein could be either reassembled into a regular array or reattached to the cell walls; RA proteins modified by the other methods lost the ability for both reassembly and reattachment. The unmodified RA protein could be reattached to periodate-oxidized cell walls, but not to methylated ones. These results suggest that the positive charge of the amino group as well as the negative charge of the carboxyl group of RA protein plays an important role(s) in morphogenesis of the hexagonal array and in its attachment to the underlying cell wall layer. The periodate-insensitive lone hydroxyl groups of the neutral polysaccharide molecule in the cell wall seem to be the receptor sites for RA protein in the attachment to the cell wall.     

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.     

Formation of non-amidine products in the chemical modification of horse liver alcohol dehydrogenase with imido esters

The reaction of imido esters with horse liver alcohol dehydrogenase (LADH) and other proteins is widely considered to involve direct conversion of amino groups to amidine functions. We have shown that the 14-fold activated form of LADH which is produced when the modification is carried out near pH 8 contains primarily N-alkyl imidate, rather than amidine, moieties. Fully acetamidinated LADH, which is formed directly at pH 10, or by multiple modification at pH 8, is 6-fold activated. The observed mechanism of amidine formation suggests a re-evaluation of various conclusions drawn from studies of protein amidination.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

Cell-cycle-dependent phosphorylation of serine and threonine in Chinese hamster cell F1 histones.

Imido esters are widely employed for the chemical modification of amino groups in proteins between pH 7–10. We have found that near pH 8 the initial products of reaction of simple primary amines with imido esters are N-alkyl imidates which subsequently react either with ammonia to yield the expected amidine or with water to form free amine. In contrast, near pH 10 amidine formation occurs more rapidly and in better yield, apparently without the accumulation of an intermediate. The observed mechanism of amidine formation implies the possible occurrence of novel side reactions and suggests improved conditions for protein amidination.     

Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).     

Selective isolation of free and blocked amino-terminal peptides from enzymatic digestion of proteins

A general method for the selective isolation of free and blocked amino-terminal peptides from proteins is described. The rationale behind the methodology is based on the reasoning that if a protein, which has all its free amino groups blocked by citraconylation, is digested with a protease, all peptides, except those derived from the amino terminus, will have a free amino group. Reaction of such a digest with 1-fluoro-2,4-dinitrobenzene (Dnp-F) followed by removal of citraconyl groups by acid treatment and removal of dinitrophenyl (Dnp) groups from histidine and tyrosine side chains by thiolysis will result in dinitrophenylation of all alpha-amino groups of peptides generated from internal cleavages, leaving only peptides derived from the amino terminus without a Dnp group. The strong adsorption of Dnp groups to polystyrene is used to selectively elute the underivatized amino-terminal peptides from such a column. It is also demonstrated how selective isolation of amino-terminal peptides can be used to determine whether a protein has a free or blocked amino terminus.     

Characterization of the reaction of methyl acetimidate with sperm whale myoglobin.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.     

Beta-hydroxyaspartic acid in vitamin K-dependent proteins

A method for the quantitation of beta-hydroxyaspartic acid in proteins is described. After hydrolysis in 6 M HCl, the beta-hydroxyaspartic acid released is quantitated on an automatic amino acid analyzer employing a pH 2.0 eluting buffer and postcolumn reaction with o-phthalaldehyde for detection. The sensitivity is about 0.01 nmol. Among vitamin K-dependent proteins, factor IX, factor X, protein C, and protein Z each contain about one residue of beta-hydroxyaspartic acid whereas protein S contains two or three residues. Prothrombin lacks beta-hydroxyaspartic acid as do a number of non-vitamin K-dependent proteins also analyzed.     

Intravital survival and deformability of weakly amidinated rat erythrocytes]

Rat erythrocytes previously labelled with 51Cr were treated with 0.1 mM and 0.5 mM dimethyladipic imidate (ADE) pH 9.5, respectively, or with borate buffer pH 9.5 for 15 minutes each. After reinjection into rats of amidinated or nonamidinated erythrocytes their elimination form the streaming blood was followed for 50 days (0.1 mM ADE) or 38 days (0.5 mM ADE). During the 1st day about 10% of amidinated erythrocytes were sequestered. There was no increased initial elimination rate of borate incubated red blood cells detectable. During the following period the elimination rate of erythrocytes amidinated with 0.5 mM ADE was slightly greater than in the case of erythrocytes treated with 0.1 mM ADE. The mean life span of 0.5 mM ADE-erythrocytes was shorter compared with the controls and with 0.1 mM ADE-erythrocytes. Red blood cells previously treated with 0.5 mM ADE are significantly less deformable. Presumably, the stiffness of amidinated erythrocytes causes their increased elimination rate.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974,Biochimie54, 907–924). When carbonylcyanide-p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide-p-trifluoromethoxyphenylhydrazone.     

Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches.

Most synthesized peptides are nowadays produced using solid-phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF-acetate) salts. However, TF-acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF-acetate exchange by another counter-ion is often required. Up to now, the classical procedure has consisted of freeze-drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa approximately 0): generally HCl (pKa = - 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF-acetate counter-ion from the dicationic octapeptide lanreotide: (i) reverse-phase HPLC, (ii) ion-exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF-acetate counter-ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF-acetate counter-ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19F-NMR, 1H-NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT-IR). We also show that ATR-IR can be used to monitor the TFA removal. The counter-ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF-acetate by any other counter-ion.     

Haloacetyl groups as reversible protection of the amino function: cleavage with 2-aminothiophenol.

Haloacetylamino acids and haloacetyl peptides react rapidly with 2-aminothiophenol in weakly alkaline media to yield 2-aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N-haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloroacetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates. Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2-haloacids to modern polypeptide chemistry are outlined.     

Methylation of ribonuclease and albumin by methyl cobalamin

The capacity of methyl cobalamine (14CH3-B12) to methylate RNase and albumin in in vitro systems was studied. Under the experimental conditions, 14CH3-B12 methylated proteins about 100--1000 times more actively than S-adenosyl-methionine, a universal donor of methyl groups. The nature of buffer and pH 2 to 8 had no noticeable effect on the methylating capacity of 14CH3-B12. However, at higher pH rate of incorporation of methyl groups slightly increased. The 14CH3-groups incorporated into RNase amino acids remained stable upon illumination, in the presence of KCN in the incubation medium, and when heated in 20% HCl for 24--72 hr at 105 degrees. Methylation did not influence the enzymic properties of RNase. The automatic amino acid analyzer showed three peaks of the label of modified amino acids in the hydrolzates of methylated RNase, one of which corresponded to methylated methionine.     

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.     

Reactions of the amino groups in ribonuclease A: II. Identification of reactive amino groups in ribonuclease

Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pK_ma. It was shown that the lys-41 amino group had a pK_a of 9.03 in TEA buffer. The pK_a values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH.     

The mimetics of antiadhesive peptides as the inhibitors of Mycobacterium kansasii phagocytosis

The alpha-guanidino acids derived of 15 proteinaceous amino acids, omega-guanidino acids with gradually increased hydrocarbon chains, and amidinated dipeptides, were tested as the mimetics of antiadhesive peptides in Mycobacteria phagocytosis inhibition. The crystal structure of omega-guanidino acids used was determined by X-ray structural analysis. It follows from our experiments that the proper distance between guanidine and carboxyl groups of effector molecules is of decisive importance for their inhibitory activity.