Cystine purification

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

An early synthesis of A5-palmitoy]-.S -[2,3-bis(palmitoyloxy)propyl]cysteine employed cysteine methyl ester, however, this leads to difficulties in the saponification step of the tri-palmitoylated residue. 96 The optimized procedure, in which the cystine di-fert-butyl ester is used, 90 is outlined in Scheme 6 after N-acylation with palmitoyl chloride, the ester is reduced to the cysteine derivative for S-alkylation with l-bromopropane-2,3-diol to yield chirally defined isomers if optically pure bromo derivatives are used. Esterification of the hydroxy groups is best carried out with a 1.25-fold excess of palmitic acid, DCC, and DMAP. The use of a larger excess of palmitoyl chloride is not recommended due to purification problems. The diastereomeric mixture can be separated by silica gel chromatography using CH2Cl2/EtOAc (20 1) as eluent and the configuration was assigned by comparison with an optically pure sample obtained with 2R)- -bromopropane-2,3-diol. 

The brain enzyme has been purified over 1000-fold and shown to be homogeneous by ultracentrifugation and electrophoresis criteria (36) the activity ratio for acetyl-P over carbamyl-P remains unchanged with purification. This enzyme is one of the smallest on record the molecular weight from physical data is 13,200 and from amino acid analysis is 12,600 the amino acid composition of the enzyme is given in Table I. The terminal amino acid is aspartic acid (25). Cystine has not been detected. 

Amino Acid Composition. Values vary substantially with differences of species, body location, and technique of isolation, purification, or analysis. However, distinctive patterns are associated with some membrane and fibrous protein isolates (Table 1). High proportions of proline and cystine are consistent with observed chemical and mechanical resistance of membranes (32, 68, 71). Similarly, increased methionine and... 

Dongjin, P. Soyoung, L. Rapid purification of micro-cystin-LR using supercritical fluid exteraction and flash chromatography. Anal. Lett. 2002, 35 (9), 1591-1602. Camel, V. Recent extraction techniques for solid matrices—Supercritical fluid extraction, pressurized fluid extraction and microwave-assisted extraction Their potential and pitfalls. Analyst 2001, 126, 1182-1193. 

The general protocol for immunoaffinity purification of the microcystins is as follows sample extracts dissolved in PBS are loaded onto the immunoaffinity column and passed through the column. Gravity flow is usually used. After washing with PBS and distilled water, the micro-cystin fraction is eluted with 100% methanol. 

Enzyme Purification. Broccoli contained sufficient levels of peroxidase, lipase and cystine lyase to permit their isolation in the amounts needed. Only traces of lipoxygenase and catalase were present. Activities (units/g vegetable see assay methods below) were peroxidase, 220 lipase, 12 lyase, 0.26. Catalase was M) units/g in broccoli compared to 19 in English green peas lipoxygenase was 2 units/g in broccoli compared to 110 in English green peas. Peroxidase, lipase and cystine lyase were purified by... 

Figure 1. Schematic diagram of the purification of peroxidase, cystine lyase and lipase from broccoli. PVP, polyvinylpyrrolidone PALP, pyridoxal-5 -phosphate.

There are at least two types of enzyme systems involved in the formation and breakage of disulfide bonds of cystine residues in proteins. A thiol-disulfide interchange enzyme (protein disulfide-isomerase EC 5.3.4.1 other name, S-S-rearrangase) was first described in 1963 ( 47) and was subsequently purified from beef liver (48,49). The molecular weight of the enzyme is 42,000. The enzyme contains three half-cystine residues, one of which must be cysteine in order for the enzyme to be active. The enzyme catalyzed the rearrangement of random incorrect pairs of half-cystine residues to the native disulfide bonds in several protein substrates. Low levels of mercaptoethanol were required for activity unless the enzyme was reduced prior to use. The efficiency of the enzyme in catalyzing the interconversion of disulfide bonds was found to be a function of the number of disulfide bonds in the substrate. Purification of a thiol-disulfide interchange enzyme from Candida claussenii has been described recently (50). 

As illustrated in Fig. 1, our approach is based on the maleimide group linked to the synthetic peptide and the thiol group placed on the protein partner. For this purpose sufficient stability of the maleimide function in the course of the peptide synthesis as well as in the final deprotection and purification step is required. A detailed analysis of these critical aspects revealed full stability of the maleimide function under the normal conditions of peptide synthesis (56.57). On the other hand, thiolated carrier proteins are readily prepared by reduction of cystine-containing proteins, by mercaptosuccinylation of carrier proteins (66,67) or by the choice of natural mono- or poly-cysteine-containing proteins (68). 

The minor fraction (25%, peak I in Fig. VII-11) was of cytoplasmic origin and had no basic character. The major fraction (75%, peak II) was of nuclear origin and was rich in arginine and half-cystine. After further purification of fraction II by chromatography on Amberlite CG-50 (2x10 cm, 0.2 M acetic acid, then 0.05 M HCl),... 

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