Peptide sulfation

A) A solution of (SMI (320 mg) in trifluoroacetic acid (7 ml) was kept under nitrogen at room temperature for 15 minutes. Ether (100 ml) was added and the precipitate filtered, washed thoroughly with ether and dried. This material (280 mg) was added to concentrated sulfuric acid (20 ml), cooled at -20°C. The solution was kept in the dry ice-acetone bath at -20°C for 75 minutes. The sulfuric acid solution was poured into ice water (80 ml). The precipitate was centrifuged, resuspended in ice water (30 ml) and 4N sodium hydroxide was added until a clear solution was obtained. After reacidification to pH 4 with dilute sulfuric acid, the precipitate formed was centrifuged, washed twice with ice water and dried. Yield 155 mg. Chromatograph of DEAE Sephadex (with ammonium carbonate buffer) yielded the desired octa-peptide sulfate ester 30 mg. 

Table 1 Amino Acid Sequences of Known Naturally Occurring Bioactive Peptides Sulfated at Tyrosine...

It was observed that for CCK and related peptides sulfation of the tyrosine residue is essential for triggering the full hormonal response.131 32 More recently, additional sulfated peptides have been identified as CCK-58, 135 phytosulfokinine-a, as peptide growth factor in plants 136 as well as various toxins from Conus venom. 137138 This observation has fostered intensive research to develop synthetic methods for efficient production of tyrosine O-sulfated peptides by chemical and enzymatic approaches. 

Amino acids, ammonium, peptides Sulfate, (sulfite)... 

In this section, we provide methods for the expression and purification of recombinant engineered TPST-1 and TPST-2 using transiently transfected HEK293-T cells. With these methods, which were adapted from published procedures (Ouyang Moore, 1998 Seibert et al., 2002), 100 pg quantities of purified TPST-1 and TPST-2 can be obtained that are sufficient for analytical- and semipreparative-scale peptide sulfation reactions (Seibert et al., 2002, 2008 Veldkamp et al., 2008, 2006). 

For certain structural and functional studies, in particular NMR spectroscopy studies, rather large quantities of sulfotyrosine peptides in the 10-mg range are required. Due to the low tyrosine sulfation activities of soluble TPST enzymes for peptide substrates, enzymatic peptide sulfation at this scale requires quantities of TPST enzymes that are difficult and expensive to obtain from transfected HEK293 cells. 

Chalinidae 300-320 300-320 Adocia, Chalinula, Haliclona, Reniera Acetylenic derivatives, macro-heterocycles, triterpenes, sulfated meroterpenes, isonitriles, aromatic carotenoids, atypical sterols, long-chain phenols, pyridine derivatives, S/N heterocycles, cyclic peptides, sulfated derivatives... 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

FIGURE 10.10 Analysis of peptides on SynChopak GPC peptide. Mobile phase 0.1 N sodium phosphate, pH 7. 0.1% sodium dodecyl sulfate. (From MICRA Scientific. Inc., with permission.)... 

In alkaline solution biuret, HN(CONH2)2 reacts with copper(II) sulfate to give a characteristic violet colour due to the formation of the complexes [Cu2(/l-OH)2(NHCONHCONH)4] (Fig. 28.6a) and [Cu(NHCONHCONH)2] . This is the basis of the biuret test in which an excess of NaOH solution is added to the unknown material together with a little CUSO4 soln a violet colour indicates the presence of a protein or other compound containing a peptide linkage. 

Nitrogen sources include proteins, such as casein, zein, lactalbumin protein hydrolyzates such proteoses, peptones, peptides, and commercially available materials, such as N-Z Amine which is understood to be a casein hydrolyzate also corn steep liquor, soybean meal, gluten, cottonseed meal, fish meal, meat extracts, stick liquor, liver cake, yeast extracts and distillers solubles amino acids, urea, ammonium and nitrate salts. Such inorganic elements as sodium, potassium, calcium and magnesium and chlorides, sulfates, phosphates and combinations of these anions and cations in the form of mineral salts may be advantageously used in the fermentation. 

Carvalho, R. S. H., Tersariol, I. L. S., Nader, H., and Nakaie, C. R., First purification of heparan sulfate disaccharides with an amine resin used as solid support for peptide synthesis, Anal. Chim. Acta, 403, 205, 2000. 

Seibert C, Cadene M, Sanhz A, Chait BT, Sakmar TP. Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence. Proc Natl Acad Sci U S A 2002 99(17) 11031— 11036. 

Tokuyama H, Ueha S, Kurachi M, et al. The simultaneous blockade of chemokine receptors CCR2, CCR5 and CXCR3 by a non-peptide chemokine receptor antagonist protects mice from dextran sodium sulfate-mediated colitis. Int Immunol 2005 17 1023-1034. 

Batinic D, Robey FA. The V3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and CD4-derived synthetic peptides. J Biol Chem 1992 267 6664-6671. 

Glucuronidation Sulfation Acetylation Peptide conjugation Glutathione conjugation... 

Hayashi, T., Nagai, Y. (1980). The anomalous behavior of collagen peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is due to the low content of hydrophobic amino acid residues. J. Biochem. (Tokyo) 87, 803-808. 

Karim, M.R., Shinagawa, S., Takagi, T. (1994). Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries. Electrophoresis 15, 1141-1146. 

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