bluuebunny/crossref_metadata_2025 huggingface dataset:
PRIMARY STRUCTURE OF THE ACTIVE TRYPTIC FRAGMENTS OF HUMAN AND MONKEY SALIVARY ANIONIC PROLINE‐RICH PROTEINS
Authors: SCHLESINGER DAVID H. , HAY DONALD I.
Published: August 2010

The primary structures of the four human salivary anionic proline‐rich proteins and an analogous protein from the saliva of a monkey (Macaca arctoides) have been further investigated. These proteins possess the unusual property of inhibiting crystal growth of calcium phosphate salts, and it has been proposed that they play an important role in the mouth, by preventing precipitation of calcium phosphate salts from the supersaturated saliva.

The tryptic fragments responsible for this activity have been isolated from all five proteins and their complete amino acid sequences determined and compared. These active fragments have been unequivocally identified as the amino‐terminal segment in all five proteins. The structures of the four human fragments are identical except for the presence of Asn at residue 4 in PRP‐1 and ‐3 instead of Asp found in PRP‐2 and ‐4. Comparison of the 30 residue human fragments with the monkey fragment shows 18 residues to be identical in these peptides, providing that residue 1 of the monkey fragment is aligned with residue 3 of the human proteins.

These studies constitute the next step in determining the mechanism of action of these unusual proteins, and in determining their minimum chain length required for inhibitory activity.
ANOMALOUS EXCHANGE KINETICS OF PEPTIDE AMIDE PROTONS IN AQUEOUS SOLUTIONS
Authors: KRAUSS ERIC M. , COWBURN DAVID
Published: August 2010

Reports concerning anomalous rates of exchange of some amides in oxytocin, alumichrome, and gramicidin S are reexamined through systematic analysis of the exchange data as a function of pH and primary structure. It is shown that such an analysis can provide useful information on secondary structure when the degree of hydrogen bonding to both the NH undergoing exchange and the neighboring carbonyl group are taken into consideration.
1H N.M.R. STUDY OF THE CONFORMATION OF [Glu 4] OXYTOCIN AND ITS LANTHANIDE COMPLEXES IN AQUEOUS SOLUTION
Authors: WALTER R. , SMITH C.W. , SARATHY K.P. , PILLAI R.P. , KRISHNA N.R. , LENKINSKI R.E. , GLICKSON J.D. , HRUBY V.J.
Published: August 2010

At 400 MHz the 1H chemical shifts, peptide NH‐CαH coupling constants (JNα) and peptide hydrogen exchange rates of [Glu4] oxytocin in aqueous solution closely resemble those previously reported for oxytocin under comparable conditions, indicating that both the parent hormone and its analogue have similar conformations in this solvent. The hydrogen exchange data suggest a dynamic equilibrium between conformation(s) in which the peptide NH's of Asn5 Cys6 are internally hydrogen bonded and conformation(s) in which these hydrogens are bonded to the solvent. [Glu4]oxytocin forms 1:1 complexes with lanthanide metal ions. The diamagnetic La3+ complex exhibits values of JNα very similar to those of the metal free hormone analogue, suggesting that coordination of the metal is accompanied by minimal perturbation of the peptide backbone. Specific average proton‐metal distances estimated from Gd3+ induced paramagnetic relaxation effects indicate that the metal is probably coordinated to the Glu4 carboxyl group and the sidechain carbonyl of Asn5. Limiting shifts induced by binding of paramagnetic Yb3+ are also reported.
TOPOCHEMICALLY RELATED HORMONE STRUCTURES: Synthesis of Partial Retro‐Inverso Analogs of LH‐RH
Authors: CHATURVEDI NISHITH , GOODMAN MURRAY , BOWERS CYRIL
Published: August 2010

The syntheses of five partial retro‐inverso luteinizing hormone‐releasing hormone (LH‐RH)analogs, [g‐Tyr5, m‐Gly6]LH‐RH, [g‐Tyr5‐r‐Gly6, R,S‐m‐Leu7]LH‐RH, [g‐p‐Glu1, m‐His2]LH‐RH, [g‐p‐Glu1 ‐r‐D‐His‐R,S‐m‐Trp3]LH‐RH, and [g‐Pro9‐ propionyl‐des‐Gly10]LH‐RH, have been accomplished by solution methods. The choice of sequence to be reversed was based on suggested biodegradation mechanisms of LH‐RH. A (gem)‐diamino alkylidene residue, which was produced via Curtius rearrangement of a peptide segment, and a 2‐substituted malonyl residue mark the initiating and terminating site, respectively, of the reversed sequence.
SEQUENCE OF THE CNBr PEPTIDE CONTAINING THE PUTATIVE ESSENTIAL TYROSINE OF BACILLUS AMYLOLIQUEFACIENS ALPHA AMYLASE*
Authors: DETERA SEVILLA D. , FRIEDBERG FELIX
Published: August 2010

The amino acid sequence of the cyanogen bromide peptide (peptide B) containing the putative essential tyrosine residue in Bacillus amyloliquefaciens alpha‐amylase (EC 3.2.1.1.) was determined. It is composed of 73 amino acids and the “active” tyrosine residue is the N‐terminus of the peptide. Upon iodination of the whole enzyme by means of a lactoperoxidase‐catalyzed reaction, a minimum of eight tyrosine residues are iodinated. Four of these belong to peptide B. Among the cyanogen bromide peptides, B is the most readily iodinated one. Hence, it is predicted that peptide B is an exposed segment of the amylase molecule.
IRREVERSIBLE THERMAL DENATURATION OF BOVINE PANCREATIC RIBONUCLEASE‐A
Authors: RAMNATH S. , VITHAYATHIL PAUL J.
Published: August 2010

The isolation and characterization of the products formed during the irreversible thermal denaturation of enzyme RNAase‐A are described. RNAase‐A, when maintained in aqueous solution at pH 7.0 and 70° for 2 h, gives soluble products which have been fractionated by gel filtration on Sephadex G‐75 into four components. These components are designated RNAase‐At1, RNAase‐At2, RNAase‐At3 and RNAase‐At4 according to the order of their elution from Sephadex G‐75. RNAase‐At4 shows the same specific activity towards yeast RNA as native RNAase‐A and is virtually indistinguishable from it by the physical methods employed. However, chromatography on CM‐cellulose separates it into three components that show the same u.v. spectra and specific activity towards yeast RNA as native RNAase‐A. RNAase‐At1, RNAase‐At2and RNAase‐At3 are all structurally altered derivatives of RNAase‐A and they exhibit low specific activity (5–10%) towards yeast RNA. In the presence of added S‐protein, all these derivatives show greatly enhanced enzymic activity. RNAase‐At1 and RNAase‐At2 are polymers, covalently crosslinked by intermolecular disulfide bridges; whereas RNAase‐At3 is a monomer. Physical studies such as 1H‐n.m.r., sedimentation analysis, u.v. absorption spectra and CD spectra reveal that RNAase‐At3 is a unfolded derivative of RNAase‐A. However, it is seen to possess sufficient residual structure which gives rise to a low but easily detectable enzymic activity.
POLY (δ‐L‐ORNITHINE)
Authors: MATHUR K.B. , PANDEY R.K. , JAGANNADHAM M.V. , BALASUBRAMANIAN D.
Published: August 2010

Poly (δ‐L‐Orn) is an example of an iso‐polypeptide (i.e. variant of the usual poly α‐peptide chain), where the α‐carboxyl and δ‐amino groups of ornithine are used in polymerization while the α‐amino groups form the side chain. A procedure for the synthesis of this iso‐polypeptide is described. Circular dichroism studies of poly (δ‐L‐Orn) and its Nα‐Boc derivative suggest that these polymers might adopt a conformation in solution similar to the β‐pleated sheet.
STUDIES ON RACEMIZATION DURING COUPLINGS USING A SERIES OF MODEL TRIPEPTIDES INVOLVING ACTIVATED RESIDUES WITH UNFUNCTIONALIZED SIDE CHAINS
Authors: BENOITON N. LEO , KURODA KEIJI
Published: August 2010

Racemization studies have been carried out using as model tests couplings of N‐benzyloxycarbonylglycyl‐l‐χ with benzyl Ne‐benzyloxycarbonyl‐l‐lysinate followed by deprotection and analysis for the diastereomeric tripeptides with an amino acid analyzer, for χ = alanine, leucine, phenylalanine, valine and isoleucine. The order of susceptibility to racemization of residues during these segment couplings depends on whether the solvent is polar or apolar, with isoleucyl and valyl, followed by phenylalanyl, being the most susceptible ones in polar solvents. The racemization depressing effect of some additives on carbodiimide‐mediated couplings has been examined. Reconciliation of apparent discrepancies in data in the literature on the relative merits of l‐hydroxy‐benzotriazole and N‐hydroxysuccinimide is made on the basis of the nature of the model tests used in acquiring the data.
SYNTHESIS OF THE DODECAPEPTIDE‐α MATING FACTOR OF SACCHAROMYCES CEREVISIAE
Authors: KHAN SHABBIR AHMED , MERKEL GLENN J. , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

The synthesis of His‐Trp‐Leu‐Gln‐Leu‐Lys‐Pro‐Gly‐Gln‐Pro‐Met‐Tyr, the dodeca‐peptide α‐mating factor from Saccharomyces cerevisiae, and its Ala2‐and Cha2‐(β‐cyclohexylalanine) analogs are reported. Peptides were synthesized in solution using a combination of mixed anhydride and 1‐hydroxybenzotriazole accelerated active ester coupling procedures. Dilute methanesulfonic acid (0.1–0.2 M) in methylene chloride‐formic acid solution was employed to specifically remove the tert.‐butoxycarbonyl group in the presence of the benzyloxycarbonyl group. Free peptides were obtained using catalytic transfer hydrogenation with formic acid as the hydrogen donor followed by mild acidolysis with trifluoroacetic acid. The α‐factor and the Cha2‐analog exhibited almost equal ability to cause “shmooing” of a‐mating types of S. cerevisiae whereas the Ala2‐analog exhibited no activity in this assay. These results differ with structure‐activity studies reported on the tridecapeptide α‐factor.
PEPTIDE SEGMENT COUPLING IN AQUEOUS MEDIUM: SILVER ION ACTIVATION OF THE THIOLCARBOXYL GROUP
Authors: BLAKE JAMES
Published: August 2010

The use of the thiolcarboxyl function for the assemblage of deblocked peptide segments in aqueous medium has been investigated. The C‐terminal thiolcarboxyl peptide Ac‐Tyr‐Arg‐Arg‐Glu‐Arg‐Gly‐SH (2a) has been synthesized by the solid‐phase method. The silver compound of peptide 2a was coupled to H‐Phe‐Ala‐Glu‐Gly‐OH in 50% aqueous dimethylformamide to give a 40% yield of Ac‐Tyr‐Arg‐Arg‐Glu‐Arg‐Gly‐Phe‐Ala‐Glu‐Gly‐OH.
β‐ENDORPHIN: SYNTHESIS AND RADIORECEPTOR BINDING ACTIVITY OF βh‐ENDORPHIN‐(1–27) AND ITS ANALOGS
Authors: ZAORAL MILAN , YAMASHIRO DONALD , HAMMONDS R. GLENN , LI CHOH HAO
Published: August 2010

βh‐Endorphin‐(1–27) (I), [Ac‐Tyr1]‐βh‐endorphin‐(1–27) (II), [Gln8]‐βh‐endor‐phin‐(1–27) (III), and [Ac‐Tyr1, Gln8]‐βh‐endorphin‐(1–27) (IV) were synthe sized by the solid‐phase method. The binding potency of I‐IV to rat brain membrane preparations was measured by radioreceptor binding assay. The relative potencies were: βh‐endorphin, 100; I, 30; II, 0.04; III, 90; IV, 0.07.
HYDROLYSIS OF SOYBEAN TRYPSIN INHIBITOR BY THE γ SUBUNIT OF 7SNGF AND EGF‐BP: Demonstration of Proteolytic Activity
Authors: GREEN DAVID A. , MOORE JOHN B.
Published: August 2010

Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF‐BP) or the γ subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF‐BP or the γ subunit of 7SNGF. The hydrolysis is pH dependent with maximum proteolysis at pH 6.0–7.0. The newly formed C‐terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2–4μg/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin‐like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event.
ISOLATION AND CHARACTERIZATION OF ALPACA GROWTH HORMONE
Authors: BONINO M.J. BISCOGLIO DE JIMENEZ , CASCONE O. , DE NUÉ A.I. ARNAO , SANTOMÉ J.A. , SÁNCHEZ D. , ORÉ R. , VILLAVICENCIO M.
Published: August 2010

A simple and mild procedure is described for the isolation of pituitary growth hormone from alpaca glands. It involves extractions in buffer at pH 8. 7 and gel filtration in Sephadex G‐75. An average yield of 1 mg purified product may be obtained from 1 g fresh alpaca pituitary glands. The alpaca hormone was exam ined for homogeneity by SDS‐polyacrylamide gel electrophoresis, gel electro‐focusing and end‐group analyses. It is a protein composed of a single polypeptide chain with phenylalanine at both ends. The C‐terminal sequence is ‐Ala‐Phe. The molecular weight is approximately 21700 and the amino acid composition very similar to that observed in equine growth hormone.
STRUCTURAL STUDIES ON YEAST 3‐PHOSPHOGLYCERATE KINASE: Linear Arrangement of the CNBr Fragments, Partial Amino Acid Sequence of the Inner Part of the Polypeptide Chain, and Analyses of the N‐Terminal Domain of the Protein
Authors: FATTOUM ABDELLATIF , ROUSTAN CLAUDE , KAROUI DALILA , FEINBERG JEANNE , PRADEL LOUISE‐ANNE , GREGOIRE JANA , ROCHAT HERVE
Published: August 2010

The purpose of this work was to contribute to the study of the covalent struc ture of yeast 3‐phosphoglycerate kinase. First, we undertook the complete align ment of the four fragments produced by cyanogen‐bromide cleavage and which constitute the intact protein; we then established the total amino acid sequence of a 30‐residue peptide and the N‐terminal sequence of a 65‐residue peptide. Second, we analyzed the acetylated state of the protein. The analyses of the acid fraction “P” obtained after digestion of 3‐phosphoglycerate kinase by pronase enabled us to determine the N‐terminal sequence of this enzyme as N‐acetylserylglycine. Third, we isolated, purified and analyzed seven tryptic peptides from a fragment containing 102 amino acids coming from the N‐terminal end of the protein. The peptides occupying the N‐ and C‐terminal ends of this fragment were also identified.
STUDIES ON HYDROGEN BONDS: Part IV. Proposed Working Criteria for Assessing Qualitative Strength of Hydrogen Bonds*
Authors: MITRA JAY ATI , RAMAKRISHNAN C.
Published: August 2010

The data obtained in the earlier parts of this series for the donor and acceptor end parameters of N‐H. O and O‐H. O hydrogen bonds have been utilised to obtain a qualitative working criterion to classify the hydrogen bonds into three categories: “very good” (VG), “moderately good” (MG) and weak (W). The general distribution curves for all the four parameters are found to be nearly of the Gaussian type. Assuming that the VG hydrogen bonds lie between 0 and ± la, MG hydrogen bonds between ± 1s̀ and ± 2s̀, W hydrogen bonds beyond ± 2s̀ (where s̀ is the standard deviation), suitable cut‐off limits for classifying the hydrogen bonds in the three categories have been derived. These limits are used to get VG and MG ranges for the four parameters 1 and θ (at the donor end) and ± and ± (at the acceptor end). The qualitative strength of a hydrogen bond is decided by the cumulative application of the criteria to all the four parameters. The criterion has been further applied to some practical examples in conformational studies such as α‐helix and can be used for obtaining suitable location of hydrogen atoms to form good hydrogen bonds. An empirical approach to the energy of hydrogen bonds in the three categories has also been presented.
THERMAL PERTURBATION SPECTROPHOTOMETRY OF LULIBERIN AND MODEL HIS‐TRP PEPTIDES
Authors: BELLO JAKE , BELLO HELENE R. , AIMOTO SABURO
Published: August 2010

Thermal perturbation (TP) spectra of luliberin were measured at pH 5–8, and compared with the model chromophores N‐Ac‐Tyr‐NH2, N‐Ac‐Trp‐NH2, t‐Boc‐His‐Trp‐NH2, H‐His‐Trp‐OH, Ac‐His‐Trp‐OH, tryptophan and cyclo‐ [His‐Trp] (all l‐isomers). Between pH 5 and neutrality, the major TP extremum of the Trp3 residue of luliberin increases by about 50%. A similar effect is seen for luliberin acetylated on Tyr5. The effect with luliberin is attributed to the protonation of the His2 residue. One proposed explanation is that the protonated imidazole orients water around the nearby indole in a different way than does unprotonated imidazole. The Tyr5 residue of luliberin behaves like N‐Ac‐Tyr‐NH2, and is considered to be well exposed to solvent. The TP spectra of N‐Ac‐Trp‐NH2, t‐Boc‐His‐Trp‐NH2, Ac‐His‐Trp‐OH, and cyclo‐[His‐Trp] are pH‐independent from pH 5 to 8. The TP spectrum of H‐His‐Trp‐NH2 has a bell‐shaped pH dependence, rising from normal at pH 3.5 to above normal at pH 6, and returning to normal at pH 8. Luliberin and model peptides show that fluorescence and TP spectra of His‐Trp sequences can respond differently to pH.
NUCLEAR MAGNETIC RESONANCE STUDY ON SOLVENT DEPENDENCE OF SIDE CHAIN CONFORMATIONS OF TYROSINE AND TRYPTOPHAN DERIVATIVES
Authors: KOBAYASHI JUN'ICHI , HIGASHIJIMA TSUTOMU , SEKIDO SACHIKO , MIYAZAWA TATSUO
Published: August 2010

The stereoselectively β‐deuterated species of Ac‐Ar‐NHMe, Ac‐Ar‐OH and Ac‐Ar‐OEt (Ar = Tyr and Trp) and H‐Trp‐NHMe were synthesized. 270‐MHz 1H n.m.r. spectra of the normal and deuterated species of these aromatic amino acid derivatives were analyzed. For most of the tyrosine and tryptophan derivatives in various polar and nonpolar solvents, the lower‐field and higher‐field β‐proton signals were found to be due to the pro‐S and pro‐R protons, respectively. However, the alternative assignments apply for H‐Trp‐NHMe in aqueous solution and for Ac‐Tyr‐NHMe and Ac‐Trp‐NHMe in nonpolar solvents. Such alternative assignments of β‐proton signals were also found for H‐Phe‐NHMe, Ac‐Phe‐NMe2 and Ac‐Phe‐OtBu. From the analyses of the 1H n.m.r. spectra of tyrosine and tryptophan derivatives, the vicinal coupling constants and rotamer populations about the Cα‐Cβ bond were determined in various solvents. The rotamer populations of H‐Trp‐NHMe, Ac‐Ar‐NHMe, Ac‐Ar‐OH and Ac‐Ar‐OEt depend significantly on solvent polarity. Each of these four types of derivatives exhibits specific solvent‐polarity dependences of rotamer populations. The solvent dependences of rotamer populations were substantially reduced on replacement of α‐carbonyl group by methylene group, suggesting the effect of this carbonyl group in the solvent dependence of rotamer populations.
DIFFERENTIAL INHIBITION OF SERINE PROTEINASES BY RABBIT α 1‐PROTEINASE INHIBITORS F AND S
Authors: KOJ A. , REGOECZI E.
Published: August 2010

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit α1‐proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly.

The number of moles of an enzyme inhibited by one mole of α1 ‐proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both α1‐proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of α1‐proteinase inhibitor without enzyme inhibition.

No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit α1‐proteinase inhibitors F and S are closely related yet functionally distinct proteins.
SOLID‐PHASE SYNTHESIS OF LOCUST ADIPOKINETIC HORMONE
Authors: YAMASHIRO DONALD , APPLEBAUM SHALOM W. , BIRIC YEHUDITH , LI CHOH HAO
Published: August 2010

The synthesis of locust adipokinetic hormone by the solid‐phase method is described. The synthetic product has been shown by bioassay to possess lipid‐mobilizing activity identical to that of the natural hormone.
EFFECT OF HYDRATION ON THE THERMAL STABILITY OF PROTEIN AS MEASURED BY DIFFERENTIAL SCANNING CALORIMETRY
Authors: FUJITA YUKIHISA , NODA YUKINAO
Published: August 2010

The thermal denaturation of chymotrypsinogen A was investigated by differential scanning calorimetry in the water‐content range 0.09–1.46 g water per g protein. At water contents above 0.82 g/g, the temperature, Td, and the enthalpy, ΔHd, of denaturation were almost independent of the water content. At lower water contents, however, both Td and ΔHd showed marked dependence on the water content. The Td increased with a decrease in the water content, whereas the ΔHd decreased with a decrease in the water content in the same region. The degree of hydration dependency of δHd exhibited a break at approximately 0.41 g/g, which indicated that at least two types of hydration phase contributed to the thermal stability of the protein. This behavior was similar to that noted for lysozyme.
REVERSIBLE MODIFICATION OF LYSINE IN β‐LACTOGLOBULIN USING CITRACONIC ANHYDRIDE
Authors: BRINEGAR A. CHRIS , KINSELLA JOHN E.
Published: August 2010

Acylation of lysine in β‐lactoglobulin‐B with citraconic anhydride resulted in a loss of free sulfhydryl groups. These were not regenerated under the conditions used to remove the modifying groups from lysine. Gel filtration and polyacryl‐ amide gel electrophoresis of the citraconylated and decitraconylated β‐lactoglobulin showed the presence of high molecular weight components. Modification of sulfhydryl groups with N‐ethylmaleimide prior to citraconylation prevented the formation of these high molecular weight components. The heterogeneity of the decitraconylated protein was attributed to a combination of inter‐molecular disulfide bonding of subunits caused by structural changes occurring during lysine modification and to alkylation of free sulfhydryl groups via the citraconyl double bond.
ISOLATION AND CHARACTERIZATION OF GONADOTROPIN ISOHORMONES FROM THE PITUITARY GLAND OF PIKE EEL (MURAENESOX CINEREUS)
Authors: HUANG FORE‐LIEN , HUANG CHANG‐JEN , LIN SUE‐HWA , LO TUNG‐BIN , PAPKOFF HAROLD
Published: August 2010

Pike eel gonadotropins were isolated from pituitary glands by 40% alcohol‐6% ammonium acetate, pH 5.1 extraction and were purified by DEAE‐cellulose chromatography and electrophoresis into four electrophoretically homogeneous forms. These four isohormones were biologically identified as gonadotropins by the stimulation of 32P‐uptake in 1‐day‐old chick testes, by the induction of ovulation in catfish, and by the in vitro production of testosterone from isolated rat Leydig cells and of androgen from carp testes. The amino acid composition of the isohormones were similar to other known piscine gonadotropins (carp and salmon) and were composed of two non‐identical subunits with Tyr and Ser as N‐terminal amino acid residues. The molecular weights of two subunits were 15 000 and 10 500, respectively, as estimated by SDS‐gel disc electrophoresis.
DISULFIDE BONDS OF GLYCOPROTEIN HORMONES: Their Selective Reduction in the β Subunits of Bovine Lutropin and Thyrotropin
Authors: REEVE JOSEPH R. , PIERCE JOHN G.
Published: August 2010

Treatment of the β subunit of either bovine lutropin (LH) or thyrotropin (TSH) at pH 8.2 by 5 mM dithioerythritol produces a set of partially reduced intermediates which, after alkylation with [14C] iodoacetate, can be separated by anion‐exchange chromatography. Fractions from LH‐β are obtained which represent molecules with 1, 2, 3 and 4 or 5 of the six disulfide bonds reduced. After full reduction, complete alkylation with non‐radioactive iodoacetate and subsequent isolation of tryptic peptides, the radioactivity of each carboxymethyl cysteine residue was determined. With LH‐β, the data show that disulfide bonds between half‐cystines 93–100 and 26–110 open sequentially and that a third bond is between half‐cystine 72 and either half‐cystine 23 or 88; a specific disulfide interchange appears to have occurred between the two last positions. As reduction proceeds, extensive interchange occurs which prevents further assignment of disulfides; the half‐cystines involved in disulfides 93–100 and 26–110, however, are not involved in the interchange. It is of interest that the assignments of bonds at 93–100 and 26–110 are in agreement with results of partial hydrolysis experiments from other laboratories, while the positions most involved in interchange are those where assignments from other laboratories differ. Under the same conditions of reduction, TSH‐β yielded fractions containing one bond open (positions 88–95, analogous to 93–100 in LH‐β) and small amounts of material in which reduction had occurred at positions 19–105 and 31–85 (analogous to 26–110 and 23–72 in LH‐β). The data indicate that during the initial steps of reduction, the β subunits of LH and TSH open in a similar fashion, though disulfide interchange may begin sooner in the case of TSH. The results strongly support the assignments of two of the six disulfides in β subunits and show that unequivocal assignment of the four remaining bonds remains to be achieved.
RAPID SEPARATION OF ENKEPHALINS AND ENDORPHINS ON SEP‐PAK® REVERSE PHASE CARTRIDGES
Authors: GAY DAVID D. , LAHTI ROBERT A.
Published: August 2010

A simple, rapid method is described for separating neuropeptides. Samples were injected on Sep‐Pak® disposable reverse phase cartridges and eluted with a step gradient of n‐propanol in formate‐pyridine buffer. Enkephalins are separated from metabolic products. Separation of α, β and γ‐endorphin is demonstrated, and Met‐ and Leu‐enkephalin are also separated. This inexpensive, reproducible method is useful in studies of neuropeptide metabolism, purification of synthesized peptides, separation prior to RIA, or concentration of neuropeptides.
USE OF PERFLUOROALKANOIC ACIDS AS VOLATILE ION PAIRING REAGENTS IN PREPARATIVE HPLC
Authors: HARDING D.R.K. , BISHOP C.A. , TARTTELIN M.F. , HANCOCK W.S.
Published: August 2010

This report describes the successful use of the ether soluble, ion pairing reagents perfluoropropionic and perfluorobutyric acid in the preparative and analytical reserved phase HPLC of underivatised peptides. The preparative separation of a l‐g sample of Pyr‐His‐Gly, the proposed anorexigenic peptide, is described on C18‐silica which was packed in a flexible‐walled cartridge and subjected to radial compression. The mobile phase consisted of an aqueous solution of perfluorobutyric acid (5 mM) and a flow rate of 100 ml/min was used. The purified peptide was simply isolated by neutralizing and freeze‐drying the corresponding peak and then extracting the excess ion pairing reagent with ether. The product was then shown to be homogeneous by analytical HPLC and amino acid analysis. The tripeptide failed to show any effect on food intake, water intake or body weight in female rats. Similarly no effect was noted on the reproductive cycles of the rat.
SEQUENCE OF CYANOGEN BROMIDE FRAGMENTS D AND E OF B. AMYLOLIQUEFACIENS ALPHA AMYLASE
Authors: Sachdev Om , Friedberg Felix
Published: August 2010

Alpha amylase (Sigma type IIA) was cleaved by cyanogen bromide. The sequences of two fragments, D which contains 88 amino acids and E which contains 56, are presented. By applying the Chou‐Fasman rules, D is predicted to have 32% beta sheet and 37% alpha helix, and E is predicted to exhibit 11% beta sheet and 71% alpha helix.
β‐ENDORPHIN: ISOLATION, AMINO ACID SEQUENCE AND SYNTHESIS OF THE HORMONE FROM HORSE PITUITARY GLANDS
Authors: Li Choh Hao , Ng Tzi Bun , Yamashiro Donald , Chung David , Hammonds R. Glenn , Tseng Liang‐Fu
Published: August 2010

Beta‐endorphin has been isolated from equine pituitaries. Its amino acid sequence is identical to that of ovine, bovine and camel β‐endorphins except for substitution of the threonine residue at position 6 by serine. The equine β‐endorphin has also been synthesized by the solid‐phase method. In comparison with the human hormone, equine β‐endorphin was shown to possess 3 times the receptor‐binding activity in rat membrane preparations and 1.6 times the analgesic potency in the mouse tail‐flick assay.
BIOLOGICAL POTENCY AND CHROMATOGRAPHIC PROPERTIES OF [ASPARTOYL3]‐SECRETIN, [β‐ASP3]‐SECRETIN, AND SECRETIN (4–27):
Authors: Voskamp D. , Beyerman H.C.
Published: August 2010

[Aspartoyl3]‐, [β‐Asp3]‐secretin and secretin (4–27) were prepared. These peptides, products of intramolecular reactions in secretin, proved to be of low biological potency. The compounds could be separated by reverse‐phase high‐ performance liquid chromatography.
PURIFICATION AND PROPERTIES OF RAT SKIN ACID PHOSPHATASES
Authors: Múkinen P.‐L. , Múkinen K.K.
Published: August 2010

Four acid phosphatases (APases I‐IV) were purified from Triton X‐100 extracts of rat skin. APase I appeared as a high‐molecular weight, most likely lysosomal lipo(glyco)‐protein with a pI of 4.3. APase I, extractable as a Triton‐protein complex with a mol.wt. of 2 106, hydrolyzed α‐naphthyl phosphate (α‐NP; Km= 0.18 mM) and p‐nitrophenyl phosphate (PNP; Km= 0.96 mM) at an almost equal rate at pH 5.0. Mo7O246‐inhibited noncompetitively (Ki= 0.15 μM). APase II had a pI of 7.8–8.0 and a mol.wt. of 104000 ± 4000. APase II is likely lysosomal and at pH 5.0 it hydrolyzed most rapidly PNP (Km= 0.057 mM), α‐NP (Km= 0.18 mM) and phenyl phosphate (PP; Km= 0.41 mM). Mo7O246‐ (Ki=2.5 nM) inhibited competitively. APase III, also of lysosomal origin, showed a pI of 5.3–5.5 and a mol.wt. of 87000 ± 3000. This enzyme, at pH 5.0, hydrolyzed most rapidly α‐NP (Km= 0.16 mM), pyridoxal 5‐phosphate (PLP; Km= 0.41 mM), PNP (Km= 0.16 mM) and PP (Km= 0.16 mM). Mo7O246‐ inhibition (Ki= 0.75 μM) was of a mixed type. p‐Chloromercuribenzoate (PCMB; Ki= 2.0 μM) inhibited noncompetitively. A pI of 5.6–6.2 and a mol.wt. of 14000 ± 400 was obtained for the cytoplasmic APase IV. This enzyme, at pH 5.0, hydrolyzed most rapidly PNP (Km= 0.16 mM), PP (Km= 0.81 mM) and riboflavin 5′‐phosphate (RP; Km= 0.20 mM). Mo7O246‐ inhibited competitively (Ki= 8.5 μM) but PCMB (Ki= 0.32 μM) showed a mixed type of inhibition. PLP may be a natural substrate of APase III and RP that of APases II and IV. The activity of APases II and III may depend on histidyl and SH‐groups, that of APase I on histidyl groups, and SH‐groups may be necessary for the activity of APase IV. All these APases seem to have an active tyrosyl and carboxyl residue. The pH dependence of the catalyses, inhibition characteristics and anatomic location showed APases I‐IV to be true skin acid phosphatases.
SYNTHESIS OF CHROMOPHORIC DEHYDRO ANALOG OF LEUCINE ENKEPHALIN
Authors: Phelps David J. , Godin James R.P.
Published: August 2010

Dehydropeptides incorporating a highly colored group have been prepared by reacting peptide esters with 4‐(4′‐dimethyl‐amino‐3′‐nitro) benzylidene‐2‐phenyloxazolin‐5‐one. This method allows a rapid synthesis of colored analogs of physiologically important peptides, in this case, leucine enkephalin.
ROLE OF NON‐PLANAR PEPTIDE UNIT IN REGULAR POLYPEPTIDE HELICES:
Authors: Nambudripad Raman , Bansal Manju , Sasisekharan V.
Published: August 2010

The effect of non‐planarity of the peptide unit on helical structures stabilized by intrachain hydrogen bonds is discussed. While the present calculations generally agree with those already reported in the literature for right‐handed helical structures, it is found that the most stable left‐handed structure is a novel helix, called the δ‐helix. Its helical parameters are close to these reported for poly‐β‐rbenzyl‐L‐aspartate. Conformational energy calculations show that poly‐β‐benzyl‐L‐aspartate with the δ‐helical structure is considerably more stable than the structure it is generally believed to take up (the ω‐helix) by about 15 kcal/mol‐residue.
USE OF THIOL ACIDS IN PEPTIDE SEGMENT COUPLING IN NON‐AQUEOUS SOLVENTS
Authors: Yamashiro Donald , Blake James
Published: August 2010

Methods for preparing protected peptide thiol acids by solution and solid‐phase procedures have been developed. The coupling of Boc‐Gly‐Ala‐SH to polymer‐bound Leu was effected with dicyclohexylcarbodiimide (DCC), 2,4‐dinitrofluorobenzene (FDNB), N‐bromosuccinimide (NBS), and heat. In each case, the resulting H‐Gly‐Ala‐Leu‐OH was analyzed for the content of H‐Gly‐D‐Ala‐Leu‐OH (Izumiya test). The sensitivity of the test (0.1% epimer) was increased to 0.003% by developing a double chromatographic procedure involving partition chromatography prior to ion‐exchange chromatography. The amounts of DL epimer found in the H‐Gly‐Ala‐Leu‐OH for the various tests were (solvent and temperature in parentheses): DCC (CH2Cl2, 0°), 0.012%; DCC (DMF, 24°), 0.04%; FDNB (DMF, 24°), 0.53%; NBS (DMF, 24°), 0.46%; heat (DMF, 60°), 0.86%. Under the same conditions conventional couplings of Boc‐Gly‐Ala‐OH gave: DCC (DMF, 24°), 16.5%; DCC/1‐hydroxybenzotriazole (DMF, 24°), 0.13%. In a competitive coupling of equimolar amounts of Boc‐Gly‐Ala‐SH and Boc‐Gly‐Ala‐OH with DCC in DMF the level of DL epimer was 0.21%, indicating that the thiol acid can be selectively coupled in the presence of carboxyl groups. In a variation of the test Boc‐Gly‐Leu‐SH coupled with DCC (CH2 Cl2, 0°) to resin bound Ala with an epimer level of 0.05%.
AMINOXY ANALOGS OF LEU‐ENKEPHALIN
Authors: Salvadori Severo , Menegatti Enea , Sarto Gianpietro , Tomatis Roberto
Published: August 2010

Seven analogs of Leu‐enkephalin where an aminoacid is replaced by an aminoxy‐acid (aminoxy‐acetic, or L‐ or D‐aminoxy‐phenylpropionic or 2‐aminoxy‐4‐methylvaleric) have been synthesized. Only analogs bearing the α‐aminoxy‐acid at position 5 have biological activity in vitro.
Acetylation of bovine growth hormone by N‐acetylimidazole
Authors: De Satz V. Blumgrund , Santome J.A.
Published: August 2010

Bovine growth hormone was chemically modified with N‐acetyhmidazole and the acetylated tyrosine residues were identified. Tyrosine 42 was the most reactive followed by tyrosines 158, 35 and 109 and to a lesser degree by tyrosines 141 and 174. Several lysine residues were also acetylated. The modified hormone retained the native α‐helix content but showed a weak growth promoting activity and was unable to compete in vivo with 125I‐labeled bovine growth hormone for the liver binding sites. The loss in growth promoting activity induced by acetylation was prevented by the amidination of lysine residues. These findings combined with results already published suggest that tyrosine residues are not important in the manifestation of the hormone growth promoting activity.
β‐Endorphin: Synthesis and properties of analogs with replacement of lysine residues by arginine
Authors: BLAKE JAMES , LI CHOH HAO , NICOLAS PIERRE
Published: August 2010

Human β‐endorphin analogs, [Arg9,19,24,28,29]‐β‐endorphin (I) and [Arg24,28,29]‐β‐endorphin (II), have been synthesized by the solid‐phase method. Peptide II had 86% of the analgesic potency and 216% of the receptor binding activity of the parent molecule. Peptide I had only 18% analgesic potency but its binding activity was more than three time greater than that of human β‐endorphin.
Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector
Authors: DARBON H. , ZLOTKIN E. , KOPEYAN C. , VAN RIETSCHOTEN J. , ROCHAT H.
Published: August 2010

The complete covalent structure of the insect toxin purified from the venom of the North‐African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a “protein” or a “peptide” program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after performic oxidation, and by partial labeling of the half cystine residues with [14C]‐iodoacetic acid and determining the specific radioactivities of the S‐[14C]‐carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half‐cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys12 in mammal toxin II is replaced by Cys38 in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half‐cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.
Chain phase trajectory of a protein molecule
Authors: VISWANADHAN V.N. , SUNDARAM K.
Published: August 2010

This paper presents the profile map of a protein conformation as a three‐dimensional e‐ø plot, where the third dimension is the residue number. The trajectories of some proteins of known crystalline structure, a few super‐secondary structures in proteins and valinomycin in conformational space are presented and examined. A graphic code for amino acids based on physico‐chemical considerations is devised as part of the present scheme. The representation presented here is useful in correlating primary structure with tertiary fold, intricate sequentially correlated structural features and for the comparison of different conformations of the same or similar polymers.
New patterns of hydrogen bonded interactions between polypeptide chains
Authors: SRIKRISHNAN T. , WINIEWICZ N. , PARTHASARATHY R.
Published: August 2010

Crystals of glycylglycylglycine (C6H11N3O4), grown from an aqueous methanol solution, are triclinic, space group P1, with the unit cell dimensions (at 22 ± 3°) a= 11.656(3), b= 14.817(3), c= 4.823(2) Å, α= 88.45(3), β= 95.96(3), γ= 105.42(3)°, Z = 4 (with two molecules in the asymmetric unit) with a density of Dobs= 1.58g·cm‐3 and Dcalc= 1.572g·cm‐3. The crystal structure was solved by a combination of multisolution and trial and error methods and refined with full‐matrix least‐squares method to a final R value of 0.036 for the observed 3021 reflections (I ≥ 2s̀). The conformation of the two molecules I and II in the asymmetric unit is very similar (except around the N‐terminal end); they have the fully extended trans‐planar conformation, and have ω values ranging from 2 to 4°. The peptide chain repeating distances (C1α ‐ C3α) are 7.27 Å and 7.18 Å in the two molecules as compared with the value of 6.68 Å for extended β‐sheets with β‐carbons. There are four different interactions between these two molecules characterized by different hydrogen bonding. Molecule I is hydrogen bonded to a neighboring molecule I using four hydrogen bonds. Molecule II is hydrogen bonded to another II, using bifurcated interactions involving the peptide nitrogen. Molecule I is hydrogen bonded to two different molecules II forming distinctly different hydrogen bonding patterns from the two mentioned above. The molecules are packed in rows, in a head‐to‐tail fashion (C‐terminal opposite N‐terminal) and are held together in sheets by hydrogen bonds between carbonyl and amide groups, corresponding to the very familiar anti‐parallel pleated sheet arrangement for polypeptides. The hydrogen bonds involving the amino nitrogens as donors are significantly longer and presumably weaker compared to those involving the NH+3 group. The C=O distances show variations that correlated with hydrogen bonding. The N‐H … O angle varies from 152 to 174° and the bent N‐H … O hydrogen bonds show bifurcated interactions.
Circular dichroism conformational study of the chemotactic peptide formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine
Authors: BAKIR M. , STEVENS E.S.
Published: August 2010

The solution circular dichroism of the chemotactic agent formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine was found to be highly solvent dependent. In relatively nonpolar solvents (fluoroalcohols) the circular dichroism is that characteristic of a folded conformation. In H2SO4 the tripeptide remains intact and displays a circular dichroism typical of highly solvated and totally disordered peptides. The order‐to‐disorder transition can be followed by addition of water to trifluoroethanol.
ROLE OF TYROSINE RESIDUES ON STRUCTURE‐FUNCTION OF FRUCTOSE‐1,6‐BIPHOSPHATE ALDOLASE FROM CERATITIS CAPITATA
Authors: GAVILANES FRANCISCO G. , GAVILANES JOSE G.
Published: August 2010

Tyrosine contributions to the structure‐function relationship in the fructose‐1, 6‐biphospate aldolase from C. capitata have been investigated. There are three well defined groups of tyrosine residues with different roles in the structure of the insect aldolase. C‐terminal tyrosine residues are essential for the maintenance of the catalytic conformation. Releasing of these residues by carboxypeptidase A treatment results in complex conformational changes according to CD studies. Another tyrosine residue group is located at the active site, and the substrate, fructose‐1,6‐biphosphate, protects it upon nitration. Chemical modification of this residue results in enzyme activity changes similar to those induced by carboxypeptidase digestion. Enzyme‐substrate interaction results in a change of the microenvironment of at least three tyrosine residues per subunit with different accessibility for tetranitromethane.
Synthesis and characterization of 1‐13C‐DLeu12,14 gramicidin A
Authors: PRASAD K. U. , TRAPANE T. L. , BUSATH D. , SZABO G. , URRY D. W.
Published: August 2010

The 13C‐D‐Leu12,14 gramicidin A was synthesized by the solid phase method incorporating 13C‐D‐leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N‐protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C‐D‐Leu12,14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1‐gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1 ‐GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1‐GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.
Isolation and properties of β‐endorphin‐(1–27)‐like peptide from bovine brains
Authors: NG TZI BUN , CHUNG DAVID , LI CHOH HAO
Published: August 2010

A β‐endorphin‐(1–27)‐like peptide was isolated from bovine cerebral hemisphere extracts by gel filtration, ion‐exchange chromatography, high performance liquid chromatography and paper electrophoresis. The peptide had tyrosine as the amino‐terminal residue and its amino acid composition was nearly identical to that of equine pituitary β‐EP‐(1–27). It had also the same mobility as equine pituitary β‐EP‐(1–27) in paper electrophoresis. In radioimmunoassay and opiate receptor‐binding assay, the brain peptide had 50% activity when compared with human β‐endorphin‐(1–27). Evidence for the occurrence of NH2‐acetylated form of β‐endorphin‐(1–27) is also presented.
Peptide formation in the presence of metal ion protecting groups
Authors: ISIED STEPHAN S. , LYON JOHN , VASSILIAN ASBED
Published: August 2010

A quantitative study of the degree of racemization induced by the [(NH3)5Co‐(III)‐] protecting group when bound to the C‐terminal of the amino acids Leu, Phe, and His, as

has been carried out. Racemization was determined by forming the diastereomeric cobalt dipeptides [(Leu)(AA)Co(III)(NH3)5] where AA = L‐Leu, L‐Phe, and L‐His; after cobalt removal (using NaBH4), the peptide diastereomers were analyzed quantitatively using an amino acid analyzer. No racemization was observed within experimental error (0.3%) as a result of the substitution of the [(NH3)5Co(III)‐] group on the amino acids and peptides studied.
Marsupial hypothalamo‐neurohypophyseal hormones
Authors: HURPET DOMINIQUE , CHAUVET MARIE‐THERESE , CHAUVET JACQUELINE , ACHER ROGER
Published: August 2010

The hypothalamo‐neurohypophyseal hormones of the brush‐tailed possum (Trichosurus vulpecula) have been purified through molecular sieving and paper chromatoelectrophoresis. Two peptides have been isolated, one with pressor property, the other with uterotonic activity. The pressor hormone has been identified as arginine vasopressin by amino acid composition and amino acid sequence. The oxytocin‐like hormone has been characterized as mesotocin by amino acid composition. The amounts per dried neuro‐intermediate gland (1.0–1.5 mg) are approximately 12 and 5 nmol, respectively.

Up to now, mesotocin has only been found in non‐mammalian tetrapods, particularly in reptiles. Its preservation in Australian marsupials (Macropodidae and Phalangeridae), and the replacement of vasotocin by a vasopressin‐like peptide, suggests that they are transition species between reptiles and Eutheria. However, the presence of mesotocin raises the question of its role in marsupial lactation.
Coordination chemistry of peptides
Authors: BRESSAN M. , ETTORRE R. , MARCHIORI F. , VALLE G.
Published: August 2010

The structure of the title compound displays a nearly planar diketopiperazine ring. The side chain is folded on the ring, the S atom making a significantly short contact with the methionine carbonyl atom. The crystal packing shows an unusual assembly of alternating layers of peptide and crystallization water molecules. The side chain sulfur atom is found to be the primary site for metal coordination of the ligand, while in some systems there is evidence of additional coordination through deprotonated amide nitrogens.
Synthesis and properties of human β‐endorphin‐(1–17) and its analogs
Authors: GARZIA RAFFAELLA , YAMASHIRO DONALD , HAMMONDS R. GLENN , LI CHOH HAO
Published: August 2010

Four analogs of human β‐endorphin (βh‐EP) were synthesized by the solid‐phase method: βh‐EP‐(1–17) (I), [D‐Ala2]‐βh‐EP‐(1–17) (II), [Gln8]‐βh‐EP‐(1–17) (III) and [D‐Ala2, Gln8]‐βh‐EP‐(1–17) (IV). Measurement in a radio‐receptor binding assay with use of tritiated βh‐EP as primary ligand gave relative potencies as follows: Met‐enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; βh‐endorphin, 2253.
Synthesis and conformational properties of a synthetic cyclic peptide for the active site of α‐chymotrypsin
Authors: SCHULTZ RICHARD M. , HUFF JOHN P. , ANAGNOSTARAS P. , OLSHER URIEL , BLOUT ELKAN R.
Published: August 2010

A nonapeptide Ac‐His‐Phe‐Gly‐Cys‐D‐Phe‐Ser‐Gly‐Glu‐Cys‐NH2 (XI) cyclized through the cysteines at positions 4 and 9 is synthesized as a model active site for the enzyme α‐chymotrypsin. A CPK model of XI indicates that the peptide will have a high probability of folding into a conformation in which the two β‐phenyls interact to form a hydrophobic site to one side of the cyclohexyl structure, and the Ser‐His‐Glu side chains form a hydrogen bonded triad over the plane of cyclopeptidyl structure. Substrates can then bind at the hydrophobic pocket formed by the β‐phenyls and be acted upon by the Ser‐His‐Glu catalytic triad, as in the enzyme. 1H n.m.r. shows: (i) multiplet peaks for the phenyl protons in D2 O that condense to a singlet in DMSO‐d6, (ii) a perturbation of the phenyl protons chemical shift on proflavin association to XI, and (iii) perturbation of the His pKa to a higher value on association of proflavin to XI. These data support the existence of a hydrophobic site and a Glu‐His interaction in the peptide. Furthermore, the > 102 better affinity of proflavin to XI than to AcTrp supports the existence of a hydrophobic site. However, no acceleration of p‐nitrophenyl acetate or trans‐cinnamoyl imidazole hydrolysis over that of imidazole is observed. The possible reasons for a lack of esterase activity in XI and other peptidyl models of serine protease active sites are discussed.