Synthetic potential of Staphylococcus aureus V8-protease: an approach toward semisynthesis of covalent analogs of alpha-chain of hemoglobin S

Enzyme-catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the alpha-chain of hemoglobin S with Staphylococcus aureus V8-protease converts the chain into a fragment-complementing system by hydrolyzing the peptide bond Glu(30)-Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of alpha-chain could be achieved if conditions for the V8-protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8-protease has been now investigated by incubating V8-protease-derived fragments of alpha-chain, namely alpha 1-30 and alpha 31-47 with the enzyme at pH 6.0 in the presence of n-propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as alpha 1-47 by amino acid analysis, redigestion with V8-protease (in the absence of n-propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of alpha 1-47 is at pH 6.0, 4 degrees C, and 24 hr of incubation with 25% n-propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of alpha 1-47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8-protease and the fact that alpha 1-30 and alpha 31-141 interact to form a native-like complex, opens up an approach for the semisynthesis of covalent analogs of alpha-chain of hemoglobin S.     

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Determination of neutral haemoglobin variants by immobilized pH gradient, reversed-phase high-performance liquid chromatography and fast-atom bombardment mass spectrometry: the case of a Hb Torino alpha 43 (CE1) Phe----Val

A neutral haemoglobin variant was identified by the combined use of different analytical methods. Isoelectric focusing on immobilized pH gradients (under denaturing conditions) allowed to detect and alpha chain variant. HPLC of tryptic digest showed that its amino-acid variation resided on T alpha 6 peptide (position 41-56). In the fast-atom bombardment (FAB) positive mass spectrum of the tryptic digest, the protonated molecular ion of the T alpha 6 peptide occurred 48 mass units lower than the normal T alpha 6 fragment, corresponding to a Phe----Val substitution. To a partial sequence determination (aminopeptidase digestion of the T alpha 6 peptide, followed by amino acid determination and FAB-MS analysis of the digestion-generated mixture) the substitution appeared to be on Phe43 (CE1). This variant is already known as Hb Torino. The procedure here described proved to be fast and simple, and feasible whenever neutral variants are supposed to occur.     

Isolation and characterization of four antibacterial peptides from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.     

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus

Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A heme binding site on myelin basic protein: characterization, location, and significance

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.     

Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.

The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of Glu-22(beta) of hemoglobin S for amidation with glucosamine

X-ray diffraction analysis of deoxyhemoglobin S crystals has implicated that a number of carboxyl groups of the protein are present at or near the intermolecular contact regions. The reactivity of these or other carboxyl groups of hemoglobin S for the amidation with an amino sugar, i.e., glucosamine, and the influence of amidation on the oxygen affinity and polymerization have been investigated. Reaction of oxyhemoglobin S at pH 6.0 and 23 degrees C with 20 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 100 mM [3H]glucosamine for 1 h resulted in an incorporation of nearly two residues of glucosamine per tetramer. The amidation was very specific for the carboxyl groups of globin; the glucosamine was not incorporated into the heme carboxyls. Derivatization of hemoglobin S by glucosamine increased the O2 affinity of the protein but had no influence on either the Hill coefficient or the Bohr effect. Amidation by glucosamine also increased the solubility of deoxyhemoglobin S by about 55%. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptides beta-T3 and beta-T5 contained the glucosamine incorporated into the protein. Sequence analysis of glucosamine-modified beta-T3 and beta-T5 demonstrated that the gamma-carboxyl groups of Glu-22 and Glu-43, respectively, had been derivatized with glucosamine. The residue Glu-43(beta) shows a high selectivity toward glycine ethyl ester also, whereas Glu-22(beta) is not reactive toward this amine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and preliminary characterization of Taenia solium metacestode proteases.

The metacestode of Taenia solium persists for years in the human central nervous system. As proteolytic enzymes play an important role in the survival of tissues helminths, we examined extracts of T. solium metacestodes for proteolytic activity using 9 synthetic peptide substrates and 3 proteins (hemoglobin, albumin, and immunoglobulin G). The proteolytic enzymes were classified based on their inhibitor profiles. At neutral pH, aminopeptidase(arginine-7-amino-4-trifluoromethylcoumarin) and endopeptidase(benzyloxy-carbonyl-glycine-glycine-arginine-7-amino-4- trifluoromethylcoumarin) substrates were cleaved. Hydrolysis of both substrates was inhibited by chelating agents, which inhibit metalloproteases. Peak activity with both substrates eluted in gel filtration fractions corresponding to a molecular weight of about 104 kDa. Cysteine protease activity was identified, which cleaved benzyloxy-carbonyl-phenylalanine-arginine-7-amino- 4-trifluoromethylcoumarin (Z-Phe-Arg-AFC) and hemoglobin. Cleavage of Z-Phe-Arg-AFC was maximal at acid pH, was stimulated by thiols, and was inhibited by leupeptin and Ep459. Peak cysteine protease activity eluted in gel filtration fractions corresponding to a molecular weight of 32 kDa. Aspartic protease activity was identified by specific inhibition with pepstatin of acid digestion of hemoglobin and immunoglobulin G. Immunoglobulin digestion occurred at acid pH, with preferential degradation of the heavy chain. Upon gel filtration chromatography, the aspartic protease activity eluted as a broad peak with maximal activity at about 90 kDa. No serine protease activity was detected. None of the parasite enzymes digested albumin. Proteolytic enzymes of T. solium may be important for parasite survival in the intermediate host, by providing nutrients and digesting host immune molecules.     

Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation

Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.     

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the rat colonic aldosterone receptor and its activation process

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.     

Product-conformation-driven ligation of peptides by V8 protease

Organic co-solvent-induced secondary conformation of alpha(17-40) of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of alpha(17-40) has been conceptualized by the general structure FR(I)-EALER-FR(II) and the pentapeptide sequence EALER playing a major role in inducing the alpha-helical conformation. The primary structure of alpha(17-40) has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated. The central pentapeptide (EALER), referred to here as splicedon,(3) appears to dictate a primary role in facilitating the splicing reaction. When the same flanking regions are used, (1) splicedons that carry amino acid residues of low alpha-helical potential, for example G at position 2 or 3 of the splicedon, generate a conformational trap of very low thermodynamic stability, giving an equilibrium yield of only 3%-5%; (2) splicedons with amino acid residues of good alpha-helical potential generate a conformational trap of medium thermodynamic stability and give an equilibrium yield of 20%-25%; (3) the splicedons with amino residues of good alpha-helical potential and also an amino acid that can generate an i, i + 4 side-chain carboxylate-guanidino (amino) interaction, a conformational trap of maximum thermodynamic stability is generated, giving an equilibrium yield of 45%-50%; and (4) the thermodynamic stability of the conformational trap of the spliced peptide is also influenced by the amino acid composition of the flanking regions. The V8 protease resistance of the spliced peptide bond is not a direct correlate of the amount of alpha-helical conformation induced into the product. The results of this study reflect the unique role of the splicedon in translating the organic co-solvent-induced product conformation as a site-specific stabilization of the spliced peptide bond. It is speculated that the splicedon with higher alpha-helical potential as compared to either one of the flanking regions achieves this by integrating its potential with that of the flanking region(s). Exchange of flanking regions with the products of other V8 protease-catalyzed splicing reactions will help to establish the general primary structural requirements of this class of splicing reactions and facilitate their application in modular construction of proteins.     

Specific modification of the carboxyl groups of hemoglobin S

The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23 degrees C for 1 h resulted in the modification of, on an average, three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7, indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the P50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide beta T5, i.e. the segment representing amino acid residues 41 to 59 of beta-chain, accounted for nearly 75% of the label associated with the globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized beta T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl group of Glu-43(beta), identifying it as the most reactive carboxyl group in hemoglobins. The results suggest that modification of the carboxyl group of hemoglobins S, presumably the gamma-carboxyl of Glu-43(beta), reduces the propensity of deoxyhemoglobin S to polymerize.     

Isolation and characterization of the bovine hypothalamic corticotropin-releasing factor

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated carboxyl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu- Leu- Asp-Ile-Ala-NH2 using approximately 650 pmol of material.     

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.     

The covalent structure of calf skin type III collagen. II. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB1,8,10,2(Positions 223--402).

The cyanogen bromide peptide alpha 1-(III)CB1,8,10,2 is 180 amino acid residues in length and occupies position 223 to 402 along the alpha 1(III) chain. In order to elucidate its amino acid sequence, alpha 1(III)CB1,8,10,2 was fragmented with hydroxylamine, protease from Staphylococcus aureus V8 and trypsin. Peptides necessary for sequence analysis with the automated Edman degradation were separated using molecular and ion exchange chromatography. Edman degradation of the hydroxylamine-derived fragments resulted in the elucidation of 80% of the entire sequence. The rest was completely established by sequence analysis of some protease V8 and trypsin-derived peptides.