Development of mild extraction methods for the analysis of natural dyes in textiles of historical interest using LC-diode array detector-MS.

Analysis of dyes extracted from textiles of historical interest can give valuable information as to where, when, and how the textiles were made. The most widely used method for extraction of colorants involves heating with HCl, which frequently decomposes glycosidic dye components to their parent aglycons, with consequent loss of information about the source of the dye. This is particularly true for flavonoid dyes, many of which are glycosides. We have developed or improved upon two mild textile extraction methods that use ethylenediaminetetraacetic acid (EDTA) and formic acid and are efficient in extracting dyes, but preserve glycosidic linkages. The relative efficiencies of the HCl, EDTA, and formic acid extraction methods are compared by analyzing extracts of dyed samples of silk using HPLC coupled with diode array and mass spectrometric detection. HPLC profiles of EDTA or formic acid extracts of silk dyed, for example, with pagoda tree buds and onionskins are clearly distinguishable as to the plant material used, whereas profiles of HCl extracts are not. Thus, extraction of textiles with EDTA or formic acid reagents can yield significantly more information about the original dyestuff than can extraction with a strong acid.

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.

Ubiquitin-activating enzyme (E1) isoforms in lens epithelial cells: origin of translation, E2 specificity and cellular localization determined with novel site-specific antibodies.

Lens development and response to peroxide stress are associated with dramatic changes in protein ubiquitination, reflecting dynamic changes in activity of the ubiquitin-activating enzyme (E1). Two isoforms of E1 (E1A and E1B) have been identified in lens cells although only one E1 mRNA, containing three potential translational start sites, has been detected. Novel, site-specific antibodies to E1 were generated and the hypothesis that the two isoforms of E1 are translated from alternative initiation codons of a single mRNA was tested. Antibodies raised against E1A-N peptide (Met(1)to Cys(23)of E1A) reacted only with E1A by immunoblot and immunoprecipitation. Antibodies raised against E1B-N peptide (Met(1)to Glu(25)of E1B or Met(41)to Glu(65)of E1A) and E1AB-C peptide (His(1030)to Arg(1058)of E1A or His(990)to Arg(1018)of E1B) reacted with both E1A and E1B. These results indicate that (1) E1A and E1B contain the same C-terminal residues; (2) E1A contains the N terminal sequence of E1B; and (3) E1B does not contain the N terminal sequence of E1A. The two isoforms of lens E1 are therefore translated from a single mRNA. Specifically, E1A is translated from the first initiation codon, and E1B translated from the second initiation codon. E1A and E1B were affinity-purified, and their ability to 'charge' ubiquitin carrier proteins (E2s) with activated ubiquitin was compared in a cell-free system. E1A and E1B were indistinguishable with respect to charging different E2s. However, E1 immunolocalization studies with human lens epithelial cells indicate that E1A and E1B are preferentially localized to the nucleus and cytosol, respectively. This observation suggests that E1A and E1B ubiquitinate different proteins and serve different functions in intact cells.

Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale.

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.

The 2.1 A structure of a cysteine protease with proline specificity from ginger rhizome, Zingiber officinale.

A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline at the P(2) position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencing and identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoprotein possessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn156. The availability of the sequence of these glycosyl chains afforded the opportunity to observe their structure and impact on protein conformation. The three-dimensional structure of GP-II has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 0.214, free R = 0.248). The overall structure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycyl endopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. The observed N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographic and noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrate residues without intervening ordered water molecules. The putative S(2) binding pocket (the proline recognition site) was identified by superposition of the GP-II structure with structures of four previously determined papain-inhibitor complexes. The particular enzymic amino acids forming the S(2) pocket of GP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelated enzymes alpha-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangement of these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular amino acids found at S(2) are consistent with a binding pocket for a moiety with the steric characteristics and charge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S(2) binding pocket of papain. The S(2) binding pocket of GP-II greatly restricts the size of the side chain which could be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain. In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolar amino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.

Artificial antifreeze polypeptides: alpha-helical peptides with KAAK motifs have antifreeze and ice crystal morphology modifying properties.

Antifreeze polypeptides from fish are generally thought to inhibit ice crystal growth by specific adsorption onto ice surfaces and preventing addition of water molecules to the ice lattice. Recent studies have suggested that this adsorption results from hydrogen bonding through the side chains of polar amino acids as well as hydrophobic interactions between the non-polar domains on the ice-binding side of antifreeze polypeptides and the clathrate-like surfaces of ice. In order to better understand the activity of one of the antifreeze polypeptide families, namely the alpha-helical type I antifreeze polypeptides, four alpha-helical peptides having sequences not directly analogous to those of known antifreeze polypeptides and containing only positively charged and non-polar side chains were synthesized. Two peptides with regularly spaced lysine residues, GAAKAAKAAAAAAAKAAKAAAAAAAKAAKAAGGY-NH2 and GAALKAAKAAAAAALKAAKAAAAAALKAAKAAGGY-NH2, showed antifreeze activity, albeit weaker than seen in natural antifreeze polypeptides, by the criteria of freezing point depression (thermal hysteresis) and ice crystal modification to a hexagonal trapezohedron. Peptides with irregular spacing of Lys residues were completely inactive. Up to now, lysine residues have not been generally associated with antifreeze activity, though they have been implicated in some antifreeze polypeptides. This work also shows that lysine residues in themselves, when properly positioned on an alpha-helical polyalanine scaffold, have all the requisite properties needed for such an activity.

Structure-function relationships in a type I antifreeze polypeptide. The role of threonine methyl and hydroxyl groups in antifreeze activity.

Several analogs of an alanine-rich, alpha-helical type I antifreeze polypeptide from the winter flounder were synthesized and studied to evaluate the role of threonine residues on antifreeze activity. In this series, the four Thr residues in the wild type polypeptide were substituted with from one to four Ser, allo-Thr, or Val residues. Circular dichroism studies determined that these substitutions did not significantly diminish alpha-helicity. Thermal hysteresis data showed that substitution of Thr by Ser resulted in moderate to complete loss of antifreeze activity, depending on the number and position of the substituted Thr residue(s). Replacement by Val, in confirmation of other recent reports, or by allo-Thr had a much less detrimental effect on activity though there were qualitative differences in activity between the mutants and the wild type AFP. Based on these results, we propose that both the methyl and hydroxyl groups of Thr, particularly of the central two Thr residues, Thr13 and Thr24, play key roles in the ice-binding properties of the antifreeze peptide. Specifically, the methyls participate in hydrophobic interactions with ice, which provide the driving force for binding and stability, whereas the hydroxyls and other polar residues control binding specificity and impart additional stability through hydrogen bonding.

Isolation and characterization of an antifreeze protein from the longhorn sculpin, Myoxocephalus octodecimspinosis.

A new type of antifreeze protein was isolated from the serum of the longhorn sculpin, Myoxocephalus octodecimspinosis, by gel filtration and high-performance liquid chromatography. This protein (LS-12) exhibits freezing point depression activity (thermal hysteresis) and ice crystal modification properties similar to those seen for other types of fish antifreeze polypeptide, except that ice crystals grow as hexagonal trapezohedra in the presence of LS-12, rather than hexagonal bipyramids usually seen. Ice crystal etching studies demonstrate that LS-12 does not bind to the hexagonal bipyramidal or secondary prism surfaces reported for the antifreeze polypeptides from winter flounder and shorthorn sculpin, respectively. Circular dichroism studies indicate that LS-12 has an alpha-helix content of about 60% at 1 degreesC, which is in good agreement with a value of about 70% predicted from the amino acid sequence. Limited proteolysis studies and further analysis of the amino acid sequence suggest that LS-12 consists of four amphipathic alpha-helices of similar length which are folded into a four-helix bundle. Based on its size (Mr=12299) and predicted tertiary structure, LS-12 can be regarded as the first example of a new class (type IV) of fish antifreeze protein.

Cloning and sequencing of cDNA encoding the LS-12 antifreeze protein in the longhorn sculpin, Myoxocephalus octodecimspinosis.

cDNA coding for an antifreeze protein (LS-12) in the longhorn sculpin, Myoxocephalus octodecimspinosis, was prepared from liver mRNA using reverse transcriptase-polymerase chain reaction coupled with 3' and 5' RACE procedures. This cDNA contains 609 base pairs, including a 384-bp open reading frame which codes for a 128-residue LS-12 precursor protein. The predicted amino acid sequence of the mature LS-12 corresponds exactly to the amino acid sequence obtained from Edman degradation [G. Deng, D.W. Andrews, R.A. Laursen, FEBS Lett., 402, 1997, pp. 17-20]. The 20 residues preceding mature LS-12 are predicted to be a signal sequence, which is presumably cleaved off before the mature, 108-residue protein is secreted into the circulatory system. This is the first report of a cDNA sequence from M. octodecimspinosis.

Amino acid sequence of a new type of antifreeze protein, from the longhorn sculpin Myoxocephalus octodecimspinosis.

A new type of fish antifreeze protein, designated here type IV, has been isolated from the longhorn sculpin, Myoxocephalus octodecimspinosis. Sequence analysis of the protein (LS-12) reveals that it contains 108 amino acids, is blocked at the N-terminus by a pyroglutamyl group and has a high (17%) content of glutamine; it is thus completely unrelated to the earlier described types I, II and III fish antifreeze proteins. Circular dichroism spectra and conformational analysis based on the sequence data indicate that LS-12 has a high helix content and probably folds as a four-helix bundle. LS-12 shows sequence similarity to certain plasma apolipoproteins known to have helix bundle structures, suggesting the possibility that LS-12 may have arisen by recruitment and mutation of a plasma apolipoprotein.

Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides.

Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha-ras, K-ras and N-ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti-ras protein monoclonal antibodies (mAbs) to the membrane-bound peptides was assessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28-39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32-35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32-38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase-activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and clinical detection of ras oncogenes in human tumor cells.

Plasminogen kringle 4 binds the heptapeptide fragment 44-50 of the plasminogen N-terminal peptide.

The interaction between the plasminogen kringle 4 module and a synthetic peptide corresponding to the tryptic heptapeptide fragment Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), segment 44-50 of the plasminogen N-terminal peptide (Wiman and Wallén, Eur J Biochem 1975; 50:489-494), has been investigated by 1H-NMR spectroscopy. AFQYHSK, as well as the shorter fragments thereof, FQYHSK, QYHSK and YHSK, all bound to kringle 4 with equilibrium association constant (Ka) values ranging between 2.5 and 8.5 mM-1. The NMR evidence also indicates that binding is mediated by the canonical kringle lysine binding site and involves the C-terminal Lys residue of the ligand peptide. The results (a) support a potential interaction between plasminogen Lys-binding kringles and the N-terminal activation peptide, and (b) unambiguously demonstrate the capability of such kringles to bind polypeptides ending with C-terminal lysine.

Nonequilibrium antifreeze peptides and the recrystallization of ice.

Evidence is presented that the nonequilibrium antifreeze peptide (AFP) from winter flounder has a special ability to inhibit recrystallization in ice only when an appreciable amount of liquid is present, as is the case when the system contains salts and the temperature is not too low. In this circumstance the AFP binds to the ice surface at the ice-solution interfaces in grain boundaries, preventing migration of the solution and effectively immobilizing the boundaries. In the absence of liquid, recrystallization inhibition appears to be a common property of many peptides. This is consistent with the view that the special effects of AFPs require a structural fit onto ice, and therefore require the AFP molecules to have the mobility to achieve that fit. Since the concentration of salt required to induce the special recrystallization inhibition effects of AFPs is lower (< 10 mM) than that found normally in physiological fluids, AFPs could play a role in the survival of organisms by preventing damage due to recrystallization. The proposition that mobility is needed for AFP molecules to produce their special influence upon ice growth argues against any special effects of AFPs in devitrification.

Amino-acid sequence of a cooperative, dimeric myoglobin from the gastropod mollusc, Buccinum undatum L.

The complete amino-acid sequence of a dimeric myoglobin from the radular mussel of the gastropod mollusc, Buccinum undatum L. has been determined. The globin, which shows cooperative binding of oxygen, contains 146 amino acids, is N-terminal aminoacetylated, and has histidine residues at position 65 and 97, corresponding to the heme-binding histidines seen in mammalian myoglobins. It shows about 75% and 50% homology, respectively, with the dimeric molluscan myoglobins from Busycon canaliculatum and Cerithidea rhizophorarum, the former of which also shows weak cooperatively, but much less similarity to other species of myoglobin and hemoglobin.

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Protease substrate specificity mapping using membrane-bound peptides.

A method is described for assessing the substrate specificity of proteases by screening for proteolytic activity against large numbers of peptides. All 400 possible peptides derived from the 20 common amino acids were synthesized on small membrane disks in the arrangement FTC-spacer-amino acid P1-amino acid P'1-spacer-membrane, where FTC is a chromophoric group. The disks are incubated simultaneously with the protease, resulting in cleavage of the peptide between the P1 and P'1 amino acids, and the absorbance of the released chromophore is measured as a function of time. As demonstrated for chymotrypsin and papain, plots of the resulting data present a perspective view of the amino acid preferences on both sides of the scissile bond. This technique is fast, requires relatively little enzyme, and can be extended to the systematic screening of longer peptides, including analogs with unnatural amino acids. It has potential use for characterizing the specificity of proteases, assessing the results of site-specific mutagenesis, and searching for optimal substrates and inhibitors.

Structure-function relationships in an antifreeze polypeptide. The role of charged amino acids.

Several analogs of an alanine-rich alpha-helical antifreeze polypeptide were synthesized and studied to evaluate the role of charged amino acids on structure and activity. alpha-Helix content and thermal stability were assessed by circular dichroism spectrometry and antifreeze activity by freezing point depression (thermal hysteresis) and ice crystal growth rate measurements. Rearrangement, deletion and replacement of charged amino acids resulted in reduced helicity and antifreeze activity in some cases, but the effects were not dramatic. We conclude that the i+4 ion pair Lys18/Glu22 helps to stabilize the alpha-helix but is not absolutely essential for activity. NH2-terminal Asp does not contribute significantly to helix stability or activity, but the COOH terminus is sensitive to modification, since replacement of Arg37 can lead to reduced helix content and activity. In general, factors which reduce alpha-helix content also reduce antifreeze activity.

Structure-function relationships in an antifreeze polypeptide. The effect of added bulky groups on activity.

We have proposed that the antifreeze activity of an alanine-rich alpha-helical antifreeze polypeptide (AFP) is due in part to side-by-side hydrophobic interactions of AFP molecules bound on the ice surface (Wen, D., and Laursen, R. A. (1992b) Biophys. J. 63, 1659-1662). To test this hypothesis and to assess the importance of a hydrophobic surface on the outward facing (non-ice-binding) portions of the helix, we synthesized several AFP analogs with up to four Ala-->Gln or Ala-->Leu replacements and determined the effect of these changes on antifreeze activity and helix stability. Although Gln replacements caused some helix destabilization and resultant reduction of antifreeze activity, they were in general well tolerated, suggesting that the bulk hydrophobicity of the non-ice-binding faces of the AFP, per se, is probably not a major factor determining AFP activity. However, placement of either Gln or Leu in position 17 on one side of the helix completely abolished activity, demonstrating that the specific location of bulky groups can dramatically alter activity. We conclude from molecular modeling studies that the effect seen by placing bulky groups in position 17 is due to steric hindrance that prevents effective association or packing of AFP molecules on the ice surface, in support of our hypothesis.

A D-antifreeze polypeptide displays the same activity as its natural L-enantiomer.

The D- and L-forms of an alpha-helical antifreeze polypeptide (AFP) have been chemically synthesized. Circular dichroism spectra of the molecules show equal and opposite ellipticites. The D- and the L-enantiomers alone, and a 50:50 mixture of the two, all show identical antifreeze activity, but the enantiomeric forms are predicted to bind to the ice surface with different orientations. It is suggested that symmetry properties of certain ice surfaces permit the asymmetric binding of AFPs, and thus that AFPs are analogous to enzymes that act upon prochiral substrates.

A model for binding of an antifreeze polypeptide to ice.

A model is proposed, based on recent peptide analog and ice crystal etching studies, whereby an alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn, and Asp side chains in a specific pattern to the [2021] hexagonal bipyramidal planes of ice. It is further suggested that this mode of binding is unidirectional, maximizing opportunities for packing of AFPs on the ice surface, and that ice crystal growth inhibition occurs by a two-step mechanism involving hydrogen bonding and hydrophobic interpeptide interactions.