4.4 The N-terminal amino acid sequence of a polypeptide chain can be easily obtained by using a fully automated sequenator 4.4.1 The machine is designed based on the edman degradation method. 4.4.2 The peptide is covalently linked to glass beads through its carboxyl terminals. One cycle of the Edman degradation is carried out in less than 2 hours 4.4.3 Usually 50(10-20)residues from the N terminal can be routinely determined by the sequenator. 4.4.4 Less than a microgram (or picomoles? of the peptide is needed for such sequence determination
4.4 The N-terminal amino acid sequence of a polypeptide chain can be easily obtained by using a fully automated sequenator. 4.4.1 The machine is designed based on the Edman degradation method. 4.4.2 The peptide is covalently linked to glass beads through its carboxyl terminals. One cycle of the Edman degradation is carried out in less than 2 hours. 4.4.3 Usually 50 (10-20) residues from the Nterminal can be routinely determined by the sequenator. 4.4.4 Less than a microgram (or picomoles?) of the peptide is needed for such sequence determination
5. Large proteins are cleaved into short peptides and then sequenced (the "divide and conquer strategy. 51 Disulfide bonds(二硫键), if exist, need to be broken first 5.1.1 The PTH-cysteines would not be released if connected by disulfide bonds 5.1.2 The disulfide bonds can be reduced by dithiothreitol(ODTT,二硫苏糖醇)orβ· mercaptoethanol(巯基乙醇), and then alkylated with iodoacetate to prevent reformation of the disulfide bonds. Addition of iodoacetate in SDS-Page can prevent cross-linking by disulfide bonds between subunits
5. Large proteins are cleaved into short peptides and then sequenced (the “divide and conquer” strategy!) 5.1 Disulfide bonds(二硫键）, if exist, need to be broken first. 5.1.1 The PTH-cysteines would not be released if connected by disulfide bonds. 5.1.2 The disulfide bonds can be reduced by dithiothreitol (DTT, 二硫苏糖醇) or bmercaptoethanol(巯基乙醇), and then alkylated with iodoacetate to prevent reformation of the disulfide bonds. Addition of iodoacetate in SDS-PAGE can prevent cross-linking by disulfide bonds between subunits
5.2 Polypeptide chains are cleaved into short fragments by chemical or enzymatic methods and then sequenced by edman method. 5.2.1 Cyanogen bromide(CNBr) cleaves polypeptides on the carboxyl side of methionine residues. 5.2.2 A set of proteases cleave peptide chains adjacent to specific amino acid residues: Trypsin, specifically on the carboxyl side ofArg(精氨酸)and Lys(赖氨酸); Chymotrypsin(胰凝乳蛋白酶,糜蛋白 酶), the carboxyl side of phe(苯丙氨酸),Tyr(酪氨 酸), and Trp(色氨酸). terminals?) 5.2.3 The fragments thus produced need to be separated( purified) by chromatographic(色谱分析的) or electrophoretic(电泳的) methods before they can be sequenced
5.2 Polypeptide chains are cleaved into short fragments by chemical or enzymatic methods and then sequenced by Edman method. 5.2.1 Cyanogen bromide (CNBr) cleaves polypeptides on the carboxyl side of methionine residues. 5.2.2 A set of proteases cleave peptide chains adjacent to specific amino acid residues: Trypsin, specifically on the carboxyl side of Arg（精氨酸）and Lys（赖氨酸）; Chymotrypsin（胰凝乳蛋白酶,糜蛋白 酶）, the carboxyl side of Phe（苯丙氨酸）, Tyr（酪氨 酸）, and Trp（色氨酸）. (terminals?) 5.2.3 The fragments thus produced need to be separated (purified) by chromatographic（色谱分析的） or electrophoretic（电泳的）methods before they can be sequenced