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Net electrical load of a peptide


I have to determine the electrical charge of the next peptide chain:

C - E - H - P

I know that this page is not there to raise doubts about this style, but I have looked for resources on the Internet and find nothing. Please explain to me how to determine the net load according to pH and the isoelectric point, so that I can take this as an example.

My teacher told me to get the necessary data from this table:


Isoelectric focusing

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). [1] [2] It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.


SENSORS | Piezoelectric Resonators

Piezoelectricity

Piezoelectricity ( piezin, Greek, to press) was first described in 1880 by Pierre and Jacques Curie, who showed that upon mechanical deformation (torsion, pressure, bending, etc.) of a solid material along an appropriate direction, electrical charges occur on the material's opposing surfaces. Conversely, applying an external electric field to a material induces a mechanical deformation. This phenomenon is called the converse piezoelectric effect. Piezoelectricity can only occur in crystals with an inversion center and from a crystallographic viewpoint, 21 point groups fulfill this requirement. However, only 20 point groups do have a nonzero piezoelectric constant. There are a large number of crystals commonly used as piezoelectric materials such as Rochelle salt, sodium chlorate, tourmaline, or quartz. Among them, α-quartz (SiO2) is very unique, as it combines mechanical, electrical, chemical, and thermal properties, which has led to its commercial significance. The quartz crystal may provide a large variety of different resonator types depending on the cut-angle with respect to the crystal lattice. The cut-angle determines the mode of induced mechanical vibration. Resonators operating in thickness shear mode (TSM), face shear mode, or flexural mode can be obtained from the mother crystal with eigenfrequencies ranging from 5×10 2 Hz to 3×10 8 Hz.


Net electrical load of a peptide - Biology



Isoelectric point, the pH at which a particular molecule carries no net electrical charge, is an critical parameter for many analytical biochemistry and proteomics techniques, especially for 2D gel electrophoresis (2D-PAGE), capillary isoelectric focusing (cIEF), X-ray crystallography and liquid chromatography–mass spectrometry (LC-MS)

  • Input should be ONE sequence in plain text or MULTIPLE sequences in FASTA format (limit is set to 50,000 chars)
  • Input should be in one letter amino acid code, input can be upper or lower case.
  • Alphabet allowed: VXCDBFMOLNYIQTGHWESKPAUR.
  • All non-amino acid characters will be removed from the sequence.
  • For big datasets use standalone version or split your input into 50k chunks.

Available also as PyPi package ( pip install isoelectric )

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Net electrical load of a peptide - Biology

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The control of ionic homeostasis in Rhodnius prolixus

The postprandial diuresis of Rhodnius

Nymphs and adults of Rhodnius prolixus are obligate blood feeders. Nymphs consume blood meals that are equivalent in mass to 10–12 times their unfed body mass, which greatly restricts their manoeuvrability, thereby making them prone to predation. A large part of the meal comprises unwanted ions (Na + and Cl – ) and water, and this excess fluid is rapidly absorbed into the haemolymph, transferred to the Malpighian tubule lumen and expelled as urine during the postprandial diuresis. The nutritious components of the meal, namely red blood cells and plasma proteins,are retained in the expanded anterior midgut, which is a functional crop, and are subsequently passed to more-posterior regions of the midgut for digestion and assimilation, which might not begin until several days after feeding(Wigglesworth, 1943). Between the infrequent blood meals, Rhodnius must conserve water and therefore does not urinate.

The rapid diuresis lasts 3–4 h, during which ∼50% of the volume load is excreted. Fig. 1presents an overview of the osmotic and ionic concentrations of fluids in different compartments of a fifth-instar Rhodnius nymph during the postprandial diuresis. Fluid is absorbed from the crop at ∼400 nl min –1 , and the volume of the crop diminishes visibly during diuresis. Fluid absorption appears to be driven by a ouabain-inhibitable Na + /K + -ATPase on the basal side (haemolymph side) of the epithelium, and the absorbate is rich in NaCl, contains little K + and is isosmotic to the blood meal, which means it is hypo-osmotic to haemolymph (Farmer et al.,1981). The volumic, osmotic and ionic challenges presented by this uptake of fluid into the haemolymph are countered by the Malpighian tubules,which remove the excess salt and water as hypo-osmotic primary urine at∼400 nl min –1 . Urine is expelled from the anus every 2–3 min, and transport processes in the hindgut have negligible impact on its volume and composition.

An overview of osmotic and ionic concentrations and fluid movements during rapid diuresis in a fifth-instar Rhodnius nymph. Coloured arrows,used to indicate transport across the anterior midgut and across upper and lower Malpighian tubule segments, correspond to those used for the dose–response curves in Fig. 4. Ion concentrations are given in mmol l –1 . Based upon data from Maddrell (Maddrell,1976).

An overview of osmotic and ionic concentrations and fluid movements during rapid diuresis in a fifth-instar Rhodnius nymph. Coloured arrows,used to indicate transport across the anterior midgut and across upper and lower Malpighian tubule segments, correspond to those used for the dose–response curves in Fig. 4. Ion concentrations are given in mmol l –1 . Based upon data from Maddrell (Maddrell,1976).

Rhodnius Malpighian tubules comprise distinct upper and lower segments. The upper segment secretes primary urine, and the rate of secretion is increased >1000-fold during the postprandial diuresis. The secreted fluid is isosmotic to haemolymph and, in unstimulated tubules, contains substantially more K + than Na + (Ramsay, 1952). During diuresis, however, Na + -rich fluid is secreted, but it nevertheless contains considerable amounts of K + (70–80 mmol l –1 ), which, if it were excreted, would deplete the haemolymph of its total K + content within a minute(Maddrell et al., 1993b). Potassium loss is prevented by the selective uptake of KCl from the lower segment of the tubule (Maddrell and Phillips, 1975). The osmotic permeability of this segment is less than that of the upper tubule, and the lower tubule is therefore a diluting segment. The urine expelled from the anus is therefore hypo-osmotic to haemolymph and contains relatively little K + (4 mmol l –1 ).

Coordinating the activities of the anterior midgut and Malpighian tubules

Within 2–3 h of the onset of feeding, a volume of NaCl-rich hypo-osmotic fluid equivalent to 10-times the haemolymph volume is absorbed from the crop of a fifth-instar nymph and expelled as urine. Despite this rapid turnover of ions and water, the volume and composition of the haemolymph change relatively little. Transport processes in the anterior midgut, and the upper and lower segments of the Malpighian tubules, must therefore be precisely coordinated. Maddrell suggested that haemolymph volume could be autonomously regulated by a diuretic hormone that stimulates Malpighian tubule secretion and fluid absorption from the anterior midgut(Maddrell, 1980). The concept is illustrated in Fig. 2. During diuresis, the diuretic hormone concentration in the circulation is somewhat greater (0–50%) than that needed to stimulate maximal tubule secretion (Maddrell, 1964a)but is assumed to be less than that required to maximally stimulate absorption from the crop. Any change in haemolymph volume will alter the diuretic hormone concentration, but will have little effect on tubule secretion, because it is already maximal. It will, however, increase or decrease the rate of absorption of fluid from the crop, thereby restoring haemolymph volume to a set point when the rates of fluid uptake and secretion are equal.

The activities of the upper and lower Malpighian tubule segments must also be closely coordinated during diuresis so as to preserve haemolymph K + . Importantly, K + uptake from the lower tubule segment must be activated in advance of the stimulation of secretion by the upper tubule so as to prevent depletion of haemolymph K + (Maddrell et al., 1993b). To achieve this, the lower tubule appears more sensitive to diuretic hormone than the upper tubule, which means it will be stimulated earlier, and its response is more rapid. During diuresis, both tubule segments are maximally stimulated,and haemolymph K + is then autonomously regulated by their differing responses to a change in K + concentration. Thus, if the haemolymph K + concentration falls, K + transport by the upper tubule decreases, whereas K + uptake from the lower tubule increases, and vice versa (Maddrell et al.,1993b).

Rhodnius diuretic hormones

Within a minute of the onset of feeding, diuretic hormone is released into the circulation, and fluid secretion by the upper tubule is stimulated>1000-fold (Maddrell,1963). The diuretic hormone originates from the mesothoracic ganglion mass (MTGM) and is released from neurohaemal sites along abdominal nerves 1–5 in response to distension of the abdomen by the blood meal(Maddrell, 1964b Maddrell, 1966). Diuretic activity is largely concentrated in a group of posterior lateral neurosecretory cells, and the hormone content of single cells isolated from the MTGM has been assessed both during and after diuresis(Berlind and Maddrell, 1979). Interestingly, these cells contain a diuretic hormone that is a potent stimulant of the upper tubule but which has no effect on K + uptake from the lower tubule, whereas that latter is stimulated by a factor releasable from the MTGM and its associated nerves, which suggests that there might be more than one diuretic hormone present in Rhodnius(Maddrell, 1976). Indeed, the MTGM is now known to contain several neurohormones that act on Malpighian tubules (Table 1). Their distribution in the MTGM is shown schematically in Fig. 3. The focus here is on the MTGM because this is the identified source of Rhodnius diuretic hormone (Maddrell, 1963), but these neurohormones are also found in other regions of the central nervous system, most notably the brain, and they might be released from the corpora cardiaca.

Diuretic and antidiuretic hormones present in the MTGM of Rhodniusand their actions

. . . Activity . . .
Hormone . Rhodnius peptide . Sequence . Crop . UMT . LMT .
Serotonin + + +
CRF-like DH (Zoone-DH) TGAVPSLSIV NPLDVLRQRL LLEIARRRMR QSQDQIQANR EMLQTI–NH2+ + 0
CT-like DH Rhopr-DH31GLDLGLSRGF SGSQAAKHLM GLAAANYAGG P–NH20 0/+ 0
Kinin (Leuma-KI) DPAFNSWG–NH20 0 0
CAP2bRhopr-CAPA-2 EGGFISFPRV–NH2 0
. . . Activity . . .
Hormone . Rhodnius peptide . Sequence . Crop . UMT . LMT .
Serotonin + + +
CRF-like DH (Zoone-DH) TGAVPSLSIV NPLDVLRQRL LLEIARRRMR QSQDQIQANR EMLQTI–NH2+ + 0
CT-like DH Rhopr-DH31GLDLGLSRGF SGSQAAKHLM GLAAANYAGG P–NH20 0/+ 0
Kinin (Leuma-KI) DPAFNSWG–NH20 0 0
CAP2bRhopr-CAPA-2 EGGFISFPRV–NH2 0

Table shows the effect of hormones on transport across the crop and upper(UMT) and lower (LMT) Malpighian tubule segments. Where the native hormone has not been identified, the sequence given is that of a peptide from the same family that has been tested for activity in Rhodnius

Activity symbols: stimulate, + inhibit, – no effect, 0. MTGM,mesothoracic ganglion mass (MTGM)

Serotonin is a diuretic hormone in Rhodnius

Of the neurohormones listed in Table 1, only serotonin (5-hydroxytryptamine, 5-HT) has been shown conclusively to function as a diuretic hormone. Serotonin-like immunoreactive material is present in dorsal unpaired medial (DUM) neurons of the MTGM(Fig. 3) and in axons extending to neurohaemal release sites along abdominal nerves 1–5(Orchard, 1989). The intensity of staining at these sites is reduced when the insect feeds, and circulating levels of serotonin are elevated within a minute of the onset of feeding(Lange et al., 1989). Moreover, the injection of 5,7-dihydroxytryptamine 24 h before feeding, which depletes nerve terminals of serotonin, either prevents or delays diuresis(Maddrell et al., 1993a).

Hypothetical dose–response curves for a diuretic hormone that stimulates fluid absorption from the anterior midgut (red curve) and fluid secretion by upper Malpighian tubules (blue curve). The vertical lines show how the diuretic hormone concentration will respond to an increase (a) or decrease (b) in haemolymph volume. Changes in diuretic hormone concentration have no effect on fluid secretion, which is already maximal, but will decrease(a) or increase (b) fluid absorption so as to restore haemolymph volume until the two rates are equal (arrow). Redrawn from Maddrell(Maddrell, 1980).

Hypothetical dose–response curves for a diuretic hormone that stimulates fluid absorption from the anterior midgut (red curve) and fluid secretion by upper Malpighian tubules (blue curve). The vertical lines show how the diuretic hormone concentration will respond to an increase (a) or decrease (b) in haemolymph volume. Changes in diuretic hormone concentration have no effect on fluid secretion, which is already maximal, but will decrease(a) or increase (b) fluid absorption so as to restore haemolymph volume until the two rates are equal (arrow). Redrawn from Maddrell(Maddrell, 1980).

Serotonin acts through cyclic AMP to maximally stimulate fluid absorption from the crop (Farmer et al.,1981), fluid secretion by the upper tubule(Maddrell et al., 1971) and K + uptake from the lower tubule(Maddrell et al., 1993b),making it an excellent candidate for a diuretic hormone that can coordinate all three activities. Normalised dose–response curves for these activities are presented in Fig. 4, along with data for the haemolymph titre of serotonin at various times before and after the onset of feeding. The circulating titre of serotonin increases from ∼7 nmol l –1 to 115 nmol l –1 within 5 min of the onset of feeding(Lange et al., 1989), which is sufficient to maximally stimulate all three target tissues(Fig. 4). Thereafter, the serotonin titre falls, and, 60 min after the onset of feeding, it is ∼20 nmol l –1 , which has no effect on the crop and upper tubule but will support ∼70% of the maximum rate of K + uptake from the lower tubule. The postprandial diuresis lasts 3–4 h, however, and this requires the release of a peptide diuretic hormone(Aston, 1979).

A schematic diagram of the posterior region of the MTGM showing the localisation of neurosecretory cells containing factors that are known to influence tubule secretion. Serotonin- and Rhopr-DH31-immunoreactive material is present in DUM neurons and axons (blue) that exit via abdominal nerves (AN) 1–5. Kinin-and CRF-like DH-immunoreactive material is present in groups of posterior lateral neurosecretory cells and axons (green) exiting via AN1 and AN2. Rhopr-CAP2b-immunoreactive material is present in three pairs of ventral medial neurosecretory cells and axons (red) exiting viaAN2–AN4. Based upon data from Orchard et al. (Orchard et al., 1989), Te Brugge et al. (Te Brugge et al.,2001 Te Brugge et al.,2005) and Paluzzi and Orchard(Paluzzi and Orchard,2006).

A schematic diagram of the posterior region of the MTGM showing the localisation of neurosecretory cells containing factors that are known to influence tubule secretion. Serotonin- and Rhopr-DH31-immunoreactive material is present in DUM neurons and axons (blue) that exit via abdominal nerves (AN) 1–5. Kinin-and CRF-like DH-immunoreactive material is present in groups of posterior lateral neurosecretory cells and axons (green) exiting via AN1 and AN2. Rhopr-CAP2b-immunoreactive material is present in three pairs of ventral medial neurosecretory cells and axons (red) exiting viaAN2–AN4. Based upon data from Orchard et al. (Orchard et al., 1989), Te Brugge et al. (Te Brugge et al.,2001 Te Brugge et al.,2005) and Paluzzi and Orchard(Paluzzi and Orchard,2006).

Candidates for the peptide diuretic hormone of Rhodnius

Possible candidates for the peptide diuretic hormone are listed in Table 1, and their localisation in neurosecretory cells of the MTGM is shown in Fig. 3. CAP2bpeptides have antidiuretic activity in Rhodnius and are dealt with separately below. Calcitonin (CT)-like diuretic hormone (CT-like DH)immunoreactivity colocalises with serotonin in DUM neurons and their neurohaemal sites. CT-like DH is therefore likely to be released into the circulation along with serotonin shortly after the onset of feeding. A CT-like DH (Rhopr-DH31) has been identified in Rhodnius(Te Brugge et al., 2008), but it has little effect on secretion by the upper tubule(Te Brugge et al., 2005) and has no effect on K + uptake from the lower tubule(Donini et al., 2008) and fluid absorption from the crop (V. A. Te Brugge and I. Orchard, personal communication).

Kinin and CRF-like peptides have not been identified in Rhodnius,but kinin-like and CRF-like immunoreactive material colocalise in 5–6 pairs of posterior lateral neurosecretory cells in the MTGM(Te Brugge et al., 2001). These are almost certainly the cells that were shown to contain a diuretic hormone that is released during diuresis(Berlind and Maddrell, 1979). Their axons extend to neurohaemal sites on abdominal nerves 1 and 2, and there is evidence to suggest that both peptides are released into the circulation in response to feeding (Te Brugge and Orchard, 2002). In cross-species assays, kinins have no effect on secretion by the upper segment of Rhodnius tubules, and this has been confirmed with HPLC fractions from the MTGM that contain kinin-like immunoreactive material (Te Brugge et al.,2002). Kinins also have no effect on K + uptake from the lower tubule (Donini et al.,2008) and fluid absorption from the crop (V. A. Te Brugge and I. Orchard, personal communication).

Normalised dose–response curves for the effects of serotonin in stimulating fluid absorption from the anterior midgut (red curve), fluid secretion by upper Malpighian tubules (blue curve) and K + uptake from lower Malpighian tubules (green curve). Vertical lines indicate serotonin concentrations in haemolymph of unfed fifth-instar Rhodnius nymphs(0), and at 5 and 60 min after the onset of feeding. Based upon data from Maddrell et al. (Maddrell et al.,1971 Maddrell et al., 1993), Farmer et al.(Farmer et al., 1981) and Lange et al. (Lange et al.,1989).

Normalised dose–response curves for the effects of serotonin in stimulating fluid absorption from the anterior midgut (red curve), fluid secretion by upper Malpighian tubules (blue curve) and K + uptake from lower Malpighian tubules (green curve). Vertical lines indicate serotonin concentrations in haemolymph of unfed fifth-instar Rhodnius nymphs(0), and at 5 and 60 min after the onset of feeding. Based upon data from Maddrell et al. (Maddrell et al.,1971 Maddrell et al., 1993), Farmer et al.(Farmer et al., 1981) and Lange et al. (Lange et al.,1989).

Cross-species assays with CRF-like DH from Locusta migratoria(Locmi-DH) and Zootermopsis nevadensis (Zoone-DH) show that both stimulate maximal secretion by the upper segment of Rhodnius tubules(Coast, 1996 Te Brugge et al., 2002). In addition, Zoone-DH stimulates fluid absorption from the crop (Te Brugge and I. Orchard, personal communication), but it has no effect on K + uptake from the lower tubule (Donini et al.,2008). The latter finding explains why the contents of isolated posterior lateral neurosecretory cells have potent diuretic activity on the upper tubule but no effect on the lower tubule(Maddrell, 1976). Zoone-DH uses cyclic AMP as a second messenger, which is consistent with what is known of the unidentified peptide diuretic hormone(Maddrell et al., 1993a), and it is likely that this is a CRF-like DH. The actions of serotonin and Zoone-DH on the upper tubule appear identical and result in a characteristic triphasic change in transepithelial potential (TEP)(Donini et al., 2008 Ianowski and O'Donnell, 2001 O'Donnell and Maddrell, 1984),which has been attributed to the sequential activation of apical membrane Cl – channels, the apical membrane V-type H + -ATPase, and a basal membrane Na + /K + /2Cl – cotransporter. The net result is a massive increase in NaCl and KCl transport into the lumen along with osmotically obliged water.

Serotonin is released rapidly into the haemolymph immediately after the onset of feeding to initiate diuresis, but the circulating titre peaks at 5 min and thereafter declines to levels below those needed to stimulate the fluid absorption from the crop and secretion by upper tubule, which would then require the release of a CRF-like DH. In support of this, the serotonin receptor antagonist ketanserin reduces the diuretic activity of haemolymph sampled 5 min after the onset of feeding by 70%, but by only 30% in samples taken after 1.5 h (Te Brugge and I. Orchard, 2002). The latter effect is considerably greater than would be anticipated, because serotonin levels have then fallen to ∼20 nmol l –1 , which have little effect on tubule secretion (see Fig. 4). The synergism demonstrated between the peptide diuretic hormone and threshold concentrations of serotonin (Maddrell et al.,1993a) could account for the marked effect of ketanserin on haemolymph diuretic activity at 1.5 h, but surprisingly neither Locmi-DH nor Zoone-DH acts synergistically with serotonin(Coast, 1996 Te Brugge et al., 2002). A different result might be obtained with native Rhodnius CRF-like DH,but synergism could not be demonstrated between serotonin and an HPLC fraction from the MTGM that contained CRF-like immunoreactive material(Te Brugge et al., 2002). As there is also no evidence of synergism between serotonin and either kinins or Rhopr-DH31, it is possible that an additional peptide diuretic hormone(s) is present in the MTGM.

Although Zoone-DH mimics serotonin in stimulating fluid secretion by the upper tubule and fluid absorption from the crop, it has no effect on K + uptake from the lower tubule(Donini et al., 2008), yet this must continue throughout diuresis to conserve haemolymph K + . This is probably achieved by the greater potency of serotonin on the lower tubule (see Fig. 4), which would allow ∼70% maximal K + uptake even when the circulating titre falls to about 20 nmol l –1 .

Terminating diuresis

Diuresis ceases 3-4 h after the onset of feeding – by when ∼50%of the imbibed salt and water have been voided. The cessation of diuresis was generally assumed to result from the removal of the stimulus for diuretic hormone release (abdominal distension) and the degradation and/or removal of diuretic hormone present in the circulation. Switching off high rates of ion and water movement across the anterior midgut and Malpighian tubules of Rhodnius by such a mechanism would be difficult, however, because at all times they must remain precisely coordinated for haemolymph volume and composition to be held constant. This might explain why Rhodnius uses an antidiuretic hormone to terminate diuresis, as first demonstrated using a CAP2b (Manse-CAP2b) from the tobacco hornworm, Manduca sexta, which acts through cyclic GMP to reduce secretion by upper tubules partially stimulated with serotonin(Quinlan et al., 1997). The native peptide, Rhopr-CAP2b (also known as Rhopr CAPA-2 because it is encoded by the Rhodnius capability gene) has subsequently been identified and shown to have potent (IC50=4 nmol l –1 ) antidiuretic activity on tubules partially stimulated by 50 nmol l –1 serotonin(Paluzzi et al., 2008). CAP2b-like immunoreactive material is present in three pairs of ventral medial neurosecretory cells in the MTGM (see Fig. 3), which express the gene encoding CAPA, and axons from these cells extend to neurohaemal areas on abdominal nerves 2–4 (Paluzzi and Orchard, 2006 Paluzzi et al.,2008). The intensity of immunoreactive staining decreases 3–4 h after the onset of feeding(Paluzzi and Orchard, 2006),which is coincident with an increase in the cyclic GMP content of Malpighian tubules in vivo (Quinlan et al.,1997) and consistent with release of Rhopr-CAP2b at the time diuresis ceases.

It has been suggested that the antidiuretic activity of CAP2bresults from the activation of a cyclic-GMP-dependent phosphodiesterase specific for cyclic AMP (Quinlan and O'Donnell, 1998), which is the second messenger used by both serotonin and the CRF-like DH. Although this is an attractive hypothesis, it has yet to be tested, but it is consistent with the observation that high concentrations of cyclic AMP reverse the effects of cyclic GMP(Quinlan and O'Donnell, 1998). Interestingly, with the addition of a high concentration (500 μmol l –1 ) of exogenous cyclic GMP, upper tubules stimulated with 10 μmol l –1 serotonin revert towards their unstimulated state by secreting K + -rich fluid(Quinlan and O'Donnell, 1998),which could be important for conserving Na + once fluid absorption from the crop ceases. The activities of the crop and the upper segment of the Malpighian tubules need to be coordinated, and recently it has been shown that Rhopr-CAP2b also reduces fluid absorption from the anterior midgut stimulated with either serotonin or Zoone-DH(Orchard and Paluzzi, 2009). The termination of diuresis in a coordinated manner would therefore depend on the potency and rate of response of the crop and upper tubule to Rhopr-CAP2b. It is not known whether Rhopr-CAP2b reduces serotonin-stimulated K + uptake from the lower tubule, where cyclic AMP is also used as a second messenger, but this might not be necessary. As diuresis ceases, K + uptake is likely to be the last process to be turned off because, while the upper tubule remains stimulated, the lower tubule must continue to reabsorb K + . Serotonin is the only diuretic hormone known to act on the lower tubule, and circulating levels at 3–4 h after the onset of feeding (∼20 nmol l –1 ) lie on the steepest part of the dose–response curve (see Fig. 4), which means that even a small decline in concentration will substantially reduce K + uptake.


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Creative Proteomics Blog

Protein sequencing is mainly relying on chemical or enzymatic digestion methods to separate peptides and detect the amount and composition of amino acid residues. This post will introduce the principles and experimental steps of protein sequencing.

Currently, the so-called protein sequencing refers to the detection of proteins’ primary structure, which contains the number of polypeptide chains in proteins. Polypeptides and proteins can be used equally in many cases. Amino acid sequence of polypeptides is the biological function of proteins.

Sequencing steps

1. Splitting polypeptide chain

Protein moleculars should be separated and purified. Several polypeptides are combined together by non-covalent bond, which is known as oligomeric protein. For example. 8 mol/L urea or 6mol / L guanidine hydrochloride can be used to deal with tetramer---Hb and dimer---Enolase.

2. Detecting the number of polypeptide in protein moleculars

The number of polypeptides can be determined by detecting the relationship between the number of moles of amino acid residues and protein molecular weight.

3. Breaking disulfide bonds

Several polypeptides chains are linked by disulfide bonds. Disulfide bonds will be reduced to thiol with excessive & [beta]- mercaptoethanol under the condition of 8mol / L urea or 6mol / L guanidine hydrochloride. And then it should be protected by alkyl reagents from re-oxidation.

Cleaving and protecting disulfide bonds
A.performic acid: -CH2SO3H
B.Reduction + oxidation: Mercaptoethanol, DTT +iodoacetic acid, -S-CH2-COOH
C.Sulfurous acid decomposition: -R1-S-S-R2 + HSO3-, R1-S- + R2-S-SOH3

4.Detecting the amino acid composition of polypeptide chains and calculating the molecular ratio of amino acid composition.


5.Sequencing N-terminal and C-terminal of polypeptide chains

Amino acid of polypeptides is divided into two categories: amino-terminal and carboxyl-terminal. The N-terminal is much more important in the analysis of amino acid sequence of peptide chains than C-terminal.

6.Polypeptide can be cleaved into several small peptides. More than two methods can be used to break peptide samples into two or more sets of peptides or peptide fragments and then separate them.

7.Determining the amino acid sequencing of each peptide

8.Determining the sequence of peptide fragments in polypeptide chains

9.Determining the position of disulfide bonds in the original polypeptide chain

Generally, pepsin will be used to deal with those polypeptide chains with disulfide bonds. And then 2D-electrophoresis technology will be used to separate each peptide fragment. Analyzing the composition and sequence of peptide fragments, which may contain disulfide bonds. And then comparing it with other peptide fragments, which are analyzed by other methods, to determine the position of disulfide bonds.

Equipped with state-of-the-art facilities, Creative Proteomics can provide protein identification services with different methods. Our team of experts with extensive experience can help you understand what you are trying to investigate and meet your requirements. Our protein sequencing services include: