disulfide cleavage

[dpar]

Hi All,

I have no experience in protein/peptide chemistry. Any help to clarify my doubts is very much appreciated.

Does the disulfide cleavage in a peptide affect the bioactivity or binding affinity ?

What if the disulfide is cleaved without affecting the active binding sites of the peptide ?

Will the conformation change affect the bioactivity/binding affinity ?

thanks
dpar

[admin]

Hello Dpar! and welcome to the forums!

Very tough and interesting question you posted. You will find that your answer depends on the protein or peptide that you are studying.

For peptides, I would believe disulfide cleavage is less likely to affect the function of a peptide, even peptides may have disulfide bonds that contribute to their structure and function.

Larger proteins depend even more on disulfide bonds, and disrupting these with beta-mercaptoethanol would damage both the structure and function of the proteins most likely. However, there may be proteins that have functional units that will not be affected by beta-mercapthoethanol. This can be found be experimentation only.

[sergbio]

Hey Dpar!

The formation of disulfide bridges is often a crucial final stage in peptide synthesis. There is compelling evidence that the disulfide pattern can be critical in the folding and structural stabilization of many natural peptide and protein sequences, while the artificial introduction of disulfide bridges into natural or designed peptides may often improve biological activities/specificities and stabilities.
Here there are some basic protocols here concerning art work with formation of disulfide bond in peptides , cleavage of S-S bond if you need to modify your peptide on Cystein edge, identification of S-S status of your protein and HPLC purification of oxidized and reduced states of peptides.

ASSAY OF FREE SULFHYDRYLS WITH ELLMAN'S REAGENT

Free sulfhydryl content of a peptide can be quantitatively determined with Ellman's reagent, 5,5-dithio-bis(2-nitrobenzoic acid). The molar extinction coefficient at 412 nm of thionitrobenzoate, the colored species generated when the reagent reacts with a free thiol, is 14,150 in 0.1 M sodium phosphate buffer The sensitivity of the reaction is in the low nmol/ml range for sulfhydryl groups, making it well suited for synthetic peptides. By dry weight most synthetic peptides are only ~60% to 75% peptide, the remainder consisting of counterions and water of hydration. Amino acid analysis is needed to establish the actual peptide content unambiguously, but such precise measurement is not usually necessary for qualitative evaluation of the free sulfhydryl content of a peptide sample.

Materials

Cysteine standard stock solution
0.1 M sodium phosphate, pH 8.0
Peptide to be assayed
Ellman's reagent solution (see recipe)
13  100-mm glass test tubes

1. Prepare a cysteine standard curve by adding 25 ul, 50 ul, 100 ul, 150 ul, 200 ul, and 250 ul of cysteine standard stock solution to separate 13  100-mm tubes. Add 250 ul of each peptide to be tested to separate tubes. Bring the volume in each tube to 250 ul with 0.1 M sodium phosphate, pH 8.0. Add 250 ul of 0.1 M sodium phosphate, pH 8.0, to a blank tube.

The cysteine content of the peptide to be assayed should fall within the range of the standard curve (37.5 to 375 nmol).

2. Add 50 ul Ellman's reagent solution and 2.5 ml of 0.1 M sodium phosphate, pH 8.0, to each tube. Mix and incubate 15 min at room temperature.

3. Measure absorbance at 412 nm (A412).

4. Plot the A412 values of the standards after subtracting the value for the blank to produce a standard curve. Use this curve to determine the free sulfhydryl content of the peptides.

Ellman's reagent solution

Dissolve 4 mg Ellman's reagent, 5,5-dithio-bis-(2-nitrobenzoic acid) (Pierce), in 1 ml of 0.1 M sodium phosphate, pH 8.0 Prepare immediately before use.


AIR OXIDATION TO A DISULFIDE

The protocol presented here is very simplistic and the complexities of correct disulfide formation in proteins may require empirically derived combinations of denaturants and reducing agents (Noiva, 1994; Chau and Nelson, 1992; Zhang and Snyder, 1991). This protocol works best for small peptides such as those obtained from peptide synthesis procedures.

Materials

Protein or peptide, lyophilized
0.1 M ammonium bicarbonate


1. Dissolve protein or peptide in 0.1 M ammonium bicarbonate.

To reduce the likelihood of intermolecular disulfide formation, keep the protein or peptide concentration 0.25 to 0.5 mg/ml.

2. Gently stir several days with vessel open to air.

3. Monitor effects of oxidation by changes in elution position on reversed-phase HPLC relative to fully reduced starting product (more sensitive for smaller peptides).

Alternatively, monitor the decrease in free sulfhydryl content colorimetrically with Ellman Reagent.

4. Desalt the oxidized protein.

ALTERNATE PROTOCOL
CHARCOAL/AIR-MEDIATED INTRAMOLECULAR DISULFIDE FORMATION

Oxygen adsorbed onto charcoal surfaces has proven efficient in mediating disulfide bond formation in a variety of peptides under basic conditions. The reactions were significantly faster than DMSO- or air-mediated cyclizations of the same substrates. Thermodynamic studies suggest that cyclization is accelerated by reduction of entropy of the peptides, upon transient adsorption to the charcoal surface, resulting in a lower activation energy (Volkmer-Engert et al., 1998).

Materials

Bis(thiol) peptide, previously purified
5% (v/v) aqueous NH4OH
Granulated charcoal

Additional reagents and equipment for assaying free sulfhydryls with Ellman's reagent
1. Dissolve the bis(thiol) peptide in water to a final concentration of 1 mg/ml (~1 mM).

2. Adjust the pH to 7.5 to 8.0 with 5% aqueous NH4OH.

3. Add granulated charcoal to the peptide solution, using up to a 1:1 (w/w) ratio of charcoal to peptide.

4. Gently shake the heterogeneous reaction mixture at 25C.

5. Monitor the progress of the reaction by Ellman's assay for disappearance of free sulfhydryls. Take 70-ul aliquots of the reaction mixture, dilute each with 0.7 ml of H2O and 70 ul of Ellman's reagent, and measure the absorbance of 2-nitro-5-thiobenzoic acid anion (NTB) at 420 nm.

In general, the reaction is complete in 2 to 6 hr.
6. Upon completion of the reaction, filter the reaction mixture, and lyophilize the filtrate.


REDUCING CYSTEINE GROUPS IN PEPTIDES

If peptides are lyophilized immediately after extraction from the resin cleavage cocktail or reversed-phase HPLC and used immediately after reconstitution, oxidation of cysteine side chains is usually not a problem. However, if oxidation to disulfides has occurred, the peptide can be reduced prior to use with the protocol presented here.

Dithiothreitol (DTT) is preferred to 2-mercaptoethanol (2-ME) as a reducing agent because its lower redox potential allows it to be effective at lower concentrations, and the reaction goes to completion because formation of the six-membered ring containing an internal disulfide is energetically favorable.

To determine if reduction is necessary, quantitate the level of free sulfhydryl groups with Ellman's reagent .
Additional methods for reducing disulfides include using sodium borohydride (Gailit, 1993) and Tris(2-carboxyethyl)phosphine (TCEP; Getz et al., 1999).

Materials

Synthetic peptide
0.1 M sodium phosphate, pH 8.0
1 M aqueous dithiothreitol (DTT)
1 N HCl
100- or 250-ul polypropylene tubes
Nitrogen gas source
Additional reagents and equipment for reversed-phase HPLC of peptides


1. Dissolve 5 to 10 mg of peptide in 0.1 M sodium phosphate, pH 8.0.

2. Add 100 ul of 1 M DTT.

3. Flush nitrogen over the surface of the liquid, seal the tube, and incubate 1 hr at 37C.

4. Acidify with 1 N HCl and desalt by reversed-phase HPLC

5. Pool peptide fractions and lyophilize. Store lyophilized at 4C until ready to use (up to several days).

The oxidation state of the peptide can usually be followed by analytical monitoring of its elution position on reversed-phase HPLC. Disulfide-linked dimers of peptides generally elute later than the monomeric peptide.


DESALTING OF PEPTIDE AND PROTEIN MIXTURES BY RP-HPLC TECHNIQUES

Introduction

RP-HPLC can be utilized to desalt peptide or protein samples derived from extraction procedures or from previous HP-HIC, HP-IEC, HP-IMAC, HP-HILIC, or HP-BAC separations. Peptide or protein solutions are injected onto a small RP-HPLC column. An aqueous buffer is used to elute the salts, while the peptides or proteins are concentrated on the top of the column. After elution of the salts, monitored by UV detection, the peptides or proteins are eluted with water-acetonitrile or water 2-propanol mobile phases. The loading capacity of an analytical column (100- to 300-mm length  4-mm i.d.) is typically ~8 mg, while the loading capacity for a semi-preparative column (30-mm length  16-mm i.d.) is ~34 mg (Pohl and Kamp, 1987).

In addition to the reagents and procedures described in the preceding sections the following materials, reagents and conditions are required:

Chromatographic Conditions

Chemicals: acetonitrile, 2-propanol, trifluoroacetic acid (TFA)

Column: e.g., C-4, C-8, C-18, etc. (10 um, 300 , 300-mm length  21.5-mm i.d.)

Sample size: 8 mg peptide or protein sample

Sample loop size: 1 ml

Step elution

Eluent A: 0.1% aqueous TFA

Eluent B: 0.1% TFA in acetonitrile or 2-propanol

Elution conditions: 100% eluent A for 3 min, then 100% eluent B for 3 min

Flow rate: 2.5 ml/min

Detection: 230 nm

Temperature: room temperature

[moleculardude]

wow great information there!