Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.     

Conformations of disulfide bridges in proteins

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of less than or equal to 2.0 A, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (phi, psi) values at Cys residues, bridge dihedral angles chi ss, chi i1, chi j1, chi i2, and chi j2, the distances C alpha i-C alpha j and C beta i-C beta j between the C alpha and C beta atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson [1981) Adv. Protein Chem. 34, 167-330). Disulfide bridging across antiparallel extended strands is observed in alpha-lytic protease, crambin, and beta-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability

Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.     

A unique lantibiotic, thermophilin 1277, containing a disulfide bridge and two thioether bridges

Aims:  To identify the chemical structure of a bacteriocin, thermophilin 1277, produced by Streptococcus thermophilus SBT1277.
Methods and Results:  Thermophilin 1277 was purified and partial N-terminal sequence analysis revealed 6 unidentified amino acids amongst 31 amino acids residues. A 2·7-kbp region containing the thermophilin 1277 structural gene ( tepA ) encoding 58 amino acids was cloned and sequenced. Mature thermophilin 1277 (33 amino acids) was preceded by a 25-amino acid putative leader peptide containing a double glycine cleavage motif. Peptide sequence analysis following chemical modification of thermophilin 1277 revealed that the Cys21 and Cys29 residues form a disulfide bridge and that Thr8 or Thr10 forms two 3-methyllanthionines with Cys13 or Cys32 via thioether bridges. Antimicrobial activity was disrupted by ethanethiol or reductive agent treatments, indicating that the internal amino acid modifications are crucial for the activity.
Conclusions:  Thermophilin 1277 from Strep. thermophilus SBT1277 belongs to the class of AII-type lantibiotics that has a disulfide and two thioether bridges.
Significance and Impact of the Study:  This is the first report of a lantibiotic produced by a GRAS species of Strep. thermophilus ; thermophilin 1277 has a unique structure containing both a disulfide bridge and two thioether bridges that are crucial for its activity.     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

The disulfide structure of mouse lysosome-associated membrane protein 1

The disulfide structure of mouse lysosome-associated membrane protein 1 has been determined by reverse-phase isolation and sequence analysis of the cysteine-containing tryptic fragments of the reduced and non-reduced deglycosylated protein. Half-cystines were distinguished (a) by their localization within tryptic or chymotryptic peptides that formed reverse-phase peaks unique to the reduced digests and (b) by their 3H-carboxymethylation only after reduction of the protein. The disulfide arrangement of the cysteines was assigned after isolation of disulfide-linked peptide pairs. Each pair chromatographed as a peak present in the nonreduced (but not the corresponding reduced) tryptic digest. NH2-terminal sequencing as well as reduction, alkylation, and rechromatography of the disulfide-linked fragments led to the following assignment of disulfide bonds: Cys11 and Cys50, Cys125 and Cys161, Cys198 and Cys235, and Cys303 and Cys340. This structure creates four 36-38-residue loops that are symmetrically placed within the two halves of the protein's intraluminal domain. The loops formed by the Cys11-Cys50 and Cys198-Cys235 bridges are homologous, and the Cys125-Cys161 and Cys303-cys340 loops form a second set of homologous domains. The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.     

Four disulfide bonds' allocation of Na+, K(+)-ATPase inhibitor (SPAI)

We have recently reported the primary structures of the three unique peptide inhibitors (SPAI-1, -2, and -3) against Na+, K(+)-ATPase which contained four disulfide bridges in common (Biochem. Biophys. Res. Commun. 164, 496 (1989)). The disulfide connectivities of SPAI were determined by the combination of amino acid analyses with the direct application to a gas-phase sequencer of its proteolytic fragments. The disulfide bond was identified by detection of phenylthiohydantoin derivatives of cystine and its decomposed product dehydroalanine. The four cysteine pairs were disclosed to be Cys20 to Cys49, Cys27 to Cys53, Cys36 to Cys48, and Cys42 to Cys57, all linked by disulfide bridge formation. The allocation pattern of these disulfide bonds was the same as that recently reported for human mucous proteinase inhibitor (EMBO J. 7, 345 (1988], though SPAI showed no proteinase inhibitory activity at all.     

Structure-receptor binding relationships of sarafotoxin and endothelin in porcine cardiovascular tissues

The specific binding of 125I-sarafotoxin S6b was observed in the microsomal fractions from porcine thoracic aorta, and two vasoconstrictive peptides with strikingly homologous structures, sarafotoxin (SRT) and endothelin (ET), interact with a common receptor of the vasculature. The order of the potency of an each endothelin or sarafotoxin analogue as a competitor against 125I-sarafotoxin S6b binding was ET-1 greater than ET-2 greater than SRT S6b greater than ET-3 much greater than SRT S6c. The hydrophobic carboxyl-terminal tail and intramolecular disulfide bridges are essential for the binding activity. In addition, Ser4, Ser5 and Lys9 seem to be important for the activity while the 6th residue does not affect the activity.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.     

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.     

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

Comparative responses to endothelin-2 and sarafotoxin 6b in the pulmonary vascular bed of the cat

Pulmonary vascular responses to endothelin-2 and sarafotoxin 6b were investigated in the feline pulmonary vascular bed under natural flow and constant flow conditions. Injections of endothelin-2 and sarafotoxin 6b in a dose of 0.3 nmol/kg iv increased pulmonary arterial and left atrial pressures and cardiac output, and caused a biphasic change in calculated pulmonary vascular resistance. Endothelin-2 caused a biphasic change in systemic arterial pressure, while sarafotoxin 6b only decreased arterial pressure. Under constant flow conditions in the intact-chest cat, injections of endothelin-2 and sarafotoxin 6b in doses of 0.1-1 nmol into the perfused lobar artery increased lobar arterial pressure in a dose-related manner but were less potent than the thromboxane A2 mimic, U46619. An ET analog with only the Cys1-Cys15 disulfide bond and an amidated carboxy terminus had no significant activity in the pulmonary vascular bed. The present data show that endothelin-2 and sarafotoxin 6b have significant vasoconstrictor activity in the pulmonary vascular bed of the cat.     

Structural characterization of the 16-kDa allergen, RA17, in rice seeds. Prediction of the secondary structure and identification of intramolecular disulfide bridges

The 16-kDa rice allergen, RA17, belonging to the alpha-amylase/trypsin inhibitor family was isolated from rice seed and structurally characterized by identifying cystine-containing peptides and predicting the secondary structure and hydrophobic regions. Eight peptides, which constitute three sets of cystine-containing peptides, were purified by HPLC from a thermolytic digest of RA17 and identified by their amino acid sequence and composition, indicating five intramolecular disulfide bridges: Cys34-Cys94, Cys26-(Cys50 or Cys51)-Cys110 and Cys12-(Cys62 or Cys64)-Cys122. Analyses of the CD spectrum and the Chou-Fasman prediction suggested that RA17 had some helical- and sheet-structure regions. Based on these experimental and predicted data, RA17 is proposed to be a globular molecule with a small hydrophobic core having folding restricted by five intramolecular disulfide bridges.     

Characterization of a recombinant murine interleukin-6: assignment of disulfide bonds

Murine interleukin 6 (mIL-6) has been synthesized as a fusion protein using a lac operon inducible plasmid in Escherichia coli. The first 8 amino acids are from the N-terminus of bacterial beta-galactosidase and the last 175 amino acids are from residue number 12 to the end of native mIL-6. This fusion protein is equipotent with the native molecule in the hybridoma growth factor assay and has comparable receptor binding characteristics. The two disulfide bridges in mIL-6 have been identified by Staphylococcus aureus V8 protease peptide mapping and Edman degradation of cystine-containing peptides. It has been shown that there are disulfide bonds between Cys46-Cys52 and Cys75-Cys85.     

Importance of the disulfide bridges in the antibacterial activity of human hepcidin

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.     

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.