Hydrophobic Moment Plot Research Articles

New User-Friendly Approach to Obtain an Eisenberg Plot and Its Use as a Practical Tool in Protein Sequence Analysis

The Eisenberg plot or hydrophobic moment plot methodology is one of the most frequently used methods of bioinformatics. Bioinformatics is more and more recognized as a helpful tool in Life Sciences in general, and recent developments in approaches recognizing lipid binding regions in proteins are promising in this respect. In this study a bioinformatics approach specialized in identifying lipid binding helical regions in proteins was used to obtain an Eisenberg plot. The validity of the Heliquest generated hydrophobic moment plot was checked and exemplified. This study indicates that the Eisenberg plot methodology can be transferred to another hydrophobicity scale and renders a user-friendly approach which can be utilized in routine checks in protein–lipid interaction and in protein and peptide lipid binding characterization studies. A combined approach seems to be advantageous and results in a powerful tool in the search of helical lipid-binding regions in proteins and peptides. The strength and limitations of the Eisenberg plot approach itself are discussed as well. The presented approach not only leads to a better understanding of the nature of the protein–lipid interactions but also provides a user-friendly tool for the search of lipid-binding regions in proteins and peptides.

A theoretical analysis of secondary structural characteristics of anticancer peptides

Here, cluster analysis showed that a database of 158 anticancer peptides formed 21 clusters based on net positive charge, hydrophobicity and amphiphilicity. In general, these clusters showed similar median toxicities (P = 0.176) against eukaryotic cell lines and no single combination of these properties was found optimal for efficacy. The database contained 14 peptides, which showed selectivity for tumour cell lines only (ACP(CT)), 123 peptides with general toxicity to eukaryotic cells (ACP(GT)) and 21 inactive peptides (ACP(I)). Hydrophobic arc size analysis showed that there was no significant difference across the datasets although peptides with wide hydrophobic arcs (>270 degrees) appeared to be associated with decreased toxicity. Extended hydrophobic moment plot analysis predicted that over 50% of ACP(CT) and ACP(GT) peptides would be surface active, which led to the suggestion that amphiphilicity is a key driver of the membrane interactions for these peptides but probably plays a role in their efficacy rather than their selectivity. This analysis also predicted that only 14% of ACP(CT) peptides compared to 45% of ACP(GT) peptides were candidates for tilted peptide formation, which led to the suggestion that the absence of this structure may support cancer cell selectivity. However, these analyses predicted that ACP(I) peptides, which possess no anticancer activity, would also form surface active and tilted alpha-helices, clearly showing that other factors are involved in determining the efficacy and selectivity of ACPs.

Oblique Orientated α-Helices and Their Prediction

Oblique orientated alpha-helices possess hydrophobicity gradients, which allow the parent alpha-helices to penetrate the membrane at a shallow angle, thereby destabilising membrane lipid organisation and promoting a range of biological processes. These alpha-helices occur in a variety of membrane interactive proteins and a number of techniques have been developed to guide their identification using sequence data alone. Hydrophobicity profiling, which provides a one-dimensional analysis of sequence data, identified only 30% of known tilted peptides in a control dataset and was thus of limited predictive use. In contrast, extended hydrophobic moment plot methodology and amphipilic profiling which take residue distribution into account and provide two-dimensional analysis of primary structural data, were found to be good indicators of tilted peptide structure. Amphiphilic profiling identified 67% of tilted peptides in the control dataset and showed that potentially, approximately 40% of transmembrane alpha-helices possess tilted peptide structure. However, it has been shown that extending these simple methods to take into account the three-dimensional spatial distribution of residues gives no clear additional benefit to identifying tilted peptides.

Sequence context and modified hydrophobic moment plots help identify `horizontal' surface helices in transmembrane protein structure prediction

Transmembrane proteins make up at least one-fifth of the genome of most organisms and are critical components of key pathways for cell survival and interactions with the environment. The function of helices found at the membrane surface in transmembrane proteins has not been greatly explored, but it is likely that they play an ancillary role to membrane spanning helices and are analogous to the surface active helices of peripheral membrane proteins, being involved in: lipid association, membrane perturbation, transmembrane signal transduction and regulation, and transmembrane helical bundle formation. Due to the difficulties in obtaining high-resolution structural data for this class of proteins, structure-from-sequence predictive methods continue to be developed as a means to obtain structural models for these largely intractable systems. A simple but effective variant of the hydrophobic moment analysis of amino acid sequences is described here as part of a protocol for distinguishing helical sequences that are parallel to or `horizontal' at the membrane bilayer/aqueous phase interface from helices that are membrane-embedded or located in extra-membranous domains. This protocol when tested on transmembrane spanning protein amino acid sequences not used in its development, was found to be 84–91% accurate when the results were compared to the partition locations in the corresponding structures determined by X-ray crystallography, and 72% accurate in determining which helices lie horizontal or near horizontal at the lipid interface.

Investigation of hydrophobic moment and hydrophobicity properties for transmembrane alpha-helices.

Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane α-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment (<μH>) and the corresponding mean hydrophobicity (<H>). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, θ, fixed at 100°.Using a data set of 403 transmembrane α-helix forming sequences, the relationship between <μH> and <H>, and the effect of varying of L and / or θ on this relationship, was investigated. Confidence intervals for correlations between <μH> and <H> are established. It is shown, using bootstrapping procedures that the strongest statistically significant correlations exist for small windows where 7 ≤ L ≤ 16. Monte Carlo analysis suggests that this correlation is dependent upon amino acid residue primary structure, implying biological function and indicating that smaller values of L give better characterisation of transmembrane sequences using <μH>. However, varying window size can also lead to different regions within a given sequence being identified as the optimal window for structure / function predictions. Furthermore, it is shown that optimal periodicity varies with window size; the optimum, based on <μH> over the range of window sizes, (7 ≤ L ≤ 16), was at θ = 102° for the transmembrane α-helix data set.

Is use of the hydrophobic moment a sound basis for predicting the structure-function relationships of membrane interactive alpha-helices?

Amphiphilic alpha-helices play a fundamental role in protein membrane association and show a segregation of polar and apolar amino acid residues. Based on correlations between amphiphilic properties and biological function, a number of theoretical approaches have been developed, which quantify alpha-helix amphiphilicity and then attempt to assign function. The most commonly used measure of amphiphilicity is the hydrophobic moment, < microH >, which, when used in conjunction with an alpha-helix's mean hydrophobicity, < H >, has been used to classify membrane interactive amphiphilic alpha-helices as either surface active or transmembrane. Here, the predictive efficacy of plot methodology is reviewed by examining published data, which compare the function of known membrane interactive amphiphilic alpha-helices to that assigned by this methodology. The results of this review are discussed in relation to the reliability of < microH > as a quantifier of alpha-helical amphiphilicity, and the ability of < microH > and < H > to describe alpha-helical structure / function relationships. It is concluded that hydrophobic moment plot methodology is not a generally reliable predictor of alpha-helical structure / function relationships. It appears that the inefficacy of plot methodology is primarily due to the inability of the plot diagram to accommodate the heterogeneity of the alpha-helical classes it attempts to define. However, the predictive efficacy of the methodology appears to be improved if other alpha-helical parameters are also considered when assigning alpha-helical function. It is suggested that the conventional methodology should be seen only as an indicator for the assignation of structure / function relationships, providing a guide to future experimental investigations.

Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide

Two membrane transporters, the 17 amino acid (aa) oligopeptide penetratin derived from the homeodomain of Antennapedia (Ant) and an analogue of the basic domain of TAT (aa 47–57) (TAT-a) from HIV-1, were tested as carriers for a p53 C-terminal peptide (aa 361–382) into human breast cancer cells. The studies were performed to determine whether the membrane-transduction efficiency of membrane carriers: Ant, TAT or TAT analogue (TAT-a) correlated with peptide hydrophobic features. Peptide-sequence analysis clearly demonstrated that the Ant sequence and p53 peptide sequence (p53p) together created a peptide with enhanced hydrophobic characteristics; while the TAT or TAT analogue (TAT-a) and p53p sequence together created a peptide with significantly less hydrophobic qualities. The degree of hydrophobic moment and helical wheel plots for these peptides correlated directly with their ability to transduce the p53 peptide. Western blot analysis revealed that Ant was able to transduce p53 C-terminal peptide into human breast cancer cells as a highly efficient membrane transporter. Compared to Ant, TAT-a fused to the C-terminus of p53 peptide (p53p–TAT-a) was a less efficient carrier into these cells under the conditions of our study. Additionally, N-terminal linked TAT-a to p53p (TAT-a–p53p) showed even lower efficiency as a transporter than p53–TAT-a. Apoptosis assays showed that the p53 peptide, fused at its C-terminus to Ant (p53p–Ant), induced a higher percentage of apoptotic cells in human breast cancer cell lines expressing mutant or wild-type p53 as compared to p53 peptide fused at its C-terminus to the TAT-a sequence (p53p–TAT-a) or when fused at the N-terminus to TAT-a (TAT-a–p53p). These data suggested a direct correlation between hydrophobic characteristics and efficiency as a transporter. Sequence study, using hydrophobic moment and helical wheel analyses, may be useful predictive tools for choosing the best carrier for a peptide.

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus, the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.

A theoretical investigation into the lipid interactions of m-calpain.

The protease, m-calpain, has been implicated in a number of pathological conditions. The enzyme is a calcium-dependent heterodimer whose activity appears to be modulated by membrane interaction involving a segment, TAMRIL, located in domain V of the protein's small subunit. Based on a sequence analysis of m-calpain, using DWIH and hydrophobic moment plot based methodologies, we have shown that this segment may contribute to a lipid interactive, oblique orientated, alpha-helical region. Our results could form a basis for future studies on the postulated lipid modulation of m-calpain activity.

Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli.

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.

Use of hydrophobic moment plot methodology to aid the identification of oblique orientated alpha-helices.

A number of alpha-helix forming peptides have been reported which appear to promote membrane fusion and other biological events related to the disruption of a hydrophobic/hydrophilic interface, due to the presence of a hydrophobicity gradient along the helical long axis. When alpha-helices from this class were analysed according to hydrophobic moment plot methodology a linear association was found to exist between the mean hydrophobic moment, <microH>, and the corresponding mean hydrophobicity, <H0>. This association was described by the least squares regression line: <microH>=0.508-0.422<H0> and, here, a methodology to aid the prediction of oblique orientated alpha-helices is presented, based on a 99% prediction band around this regression line. This methodology is intended to provide an initial identification of candidates for further investigation by other techniques such as the molecular hydrophobic potential and laboratory based experimentation, not to assign function.

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41 000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519–541), and also FP analogs (FP520V/E with Val→Glu at residue 520; FP527L/R with Leu→Arg at 527; FP529F/Y with Phe→Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.

Sequence analysis of Pasteurella multocida major outer membrane protein (OmpH) and application of synthetic peptides in vaccination of chickens against homologous strain challenge

Pasteurella multocida major outer membrane protein (OmpH) has been previously characterized as a porin. The native OmpH from strain X-73 (serotype 1) but not recombinant protein from Escherichia coli induced homologous protection in chickens. In this study OmpH sequences from 15 P. multocida serotypes as well as the CU vaccine strain were compared by sequence alignment and revealed high homology, with major variations confined to two discrete regions which were correspondingly predicted as two largest external loops. Secondary structures of OmpHs were predicted by sequence alignment of OmpHs with well defined porins and analyses of amphiphilicity, hydrophobic moment and antigenic index plots. Several synthetic peptides derived from predicted loop 2 and loop 5 of X-73 OmpH were synthesized as vaccine candidates. Vaccination studies in chickens showed that the cyclic synthetic peptide (Cyclic-L2) mimicking the predicted loop 2 induced 70% protection in chickens against strain X-73 challenge. This is the first report that a synthetic peptide mimicking the conformational epitopes of a native protein provide practical protection in target animal against bacterial infection.

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a β-strand/loop/β-strand structure secured by hydrophobic interactions and predictive of activity

A novel fragment of chromogranin A, known as `catestatin' (bovine chromogranin A 344–364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29–35.5% identity, general alignment score 44–57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, 1PKM, and 2IG2. Each of these sequences exists in nature as a β-strand/loop/β-strand structure, stabilized by hydrophobic interactions between the β-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic β-strand. Circular dichroism in the far ultraviolet showed substantial (63%) β-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg 351, Arg 353, and Arg 358), though not for two arginine residues in the strand region toward the amino-terminus. [ 125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was `docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the β and δ subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.

Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

An algorithm for the detection of surface-active α helices with the potential to anchor proteins at the membrane interface

Surface-active peptides are amphiphilic in nature and have been shown to have the potential to interact at the membrane interface, possibly by lying parallel to the membrane surface. Present methodology for the identification of these helices uses a fixed window size, is based on a two-dimensional sum of hydrophobicity vectors and gives no measure of the statistical significance for any region identified as amphiphilic. Identification of weakly surface-active structures is difficult and here we have attempted to remedy this by introducing an algorithm which considers three-dimensional geometries and variable window size. A new measure of membrane-interactive potential is proposed, called the depth-weighted inserted hydrophobicity (DWIH), which is based on the sequestration of hydrophobic residues within a hydrophobic compartment, such as that produced by a membrane bilayer. A statistical significance for this measure has been derived using Monte Carlo techniques. The algorithm is applied to a set of proteins which are thought to anchor to the membrane via C-terminal amphiphilic alpha helices. The DWIH measure appears to allow the identification of this category of membrane-interactive helices which lie near the boundary of the hydrophobic moment plot and which have previously been hard to classify.

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic α helix

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic α helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne ( Von Heijne, G. (1992) J. Mol. Biol. 225, 487–494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH 2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH 2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic α helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% α helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (≫ 250 μM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Identification of a second membrane-active 13-residue peptide segment in the antimicrobial protein, bovine seminalplasmin

Seminalplasmin (SPLN) is a 47-residue protein from bovine seminalplasma having broad-spectrum antibacterial activity. The protein has no hemolytic activity. SPLN interacts with lipid vesicles and its antibacterial activity appears to stem from its ability to permeabilize the bacterial plasma membrane. Analysis of SPLN's primary structure, with respect to its relative hydrophobicity and hydrophilicity, revealed a segment, PKLLETFLSKWIG, more hydrophobic than the rest of the protein. A synthetic peptide corresponding to this region had not only antibacterial activity but also hemolytic properties. Analysis of the SPLN sequence based on hydrophobic moment plots has revealed a second segment, SLSRYAKLANRLA, which could be membrane active. A synthetic peptide corresponding to this region shows only antibacterial activity with no hemolytic activity.

A topological model for hepatitis B surface antigen.

A model of hepatitis B surface antigen has been derived, based on extensive sequence analysis and biochemical data. The surface antigen sequences of the human, woodchuck, ground squirrel and duck hepadnaviruses were examined using hydrophobicity, hydrophobic moments, flexibility and secondary structure prediction. The helix phase diagram, which is a modified version of Eisenberg's hydrophobic moment plots and which specifically addresses the problem of transmembrane helices, was used to examine the predicted helices. In this model four transmembrane helices are predicted. The N and C termini and the second hydrophilic region, which bears the major B-cell antigenic determinants, are external. It is suggested that the transmembrane helices may pack to form a channel through the membrane and may also be involved in the mechanisms of cell entry. A significant difference between the duck hepadnavirus and the mammalian HBsAg sequences was found, hence care must be taken when extrapolating data between the duck and the human surface antigen.

Secondary structures of rat lipolytic enzymes: Circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of α-helices, β-sheets, and random coils. Lingual lipase had an ellipticity of −20235±140 deg cm 2/dmol(mean±SE) at 220nm suggesting 60% α-helix, 20% β-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was −14093±82 deg cm 2/dmol (mean ±SE) at 210nm suggesting a 34.8% α-helical, 25% β-sheet and 40% random coil secondary structure. An α-helical stretch of residues with a large hydrophobic moment (“globular” α-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.