Biochemistry-chapter 3 AA and primary structure of protein(p

3.7 Amino Acid Composition of Proteins 3.8 Determining the Sequence of Amino Acid residues

3.7 Amino Acid Composition of ProteinsHydrolyzation of proteins:

complete Hydrolyzationincomplete Hydrolyzation acid Hydrolyzation

AAsAAs+peptides

Methods

basic Hydrolyzationenzyme Hydrolyzation

1. Separation of amino acids by ion exchange chromatography

Amino Acid Separation

Unfortunately, amino acids are not colored as described in this overhead. Therefore, what methods would you use to first check if an amino acid is indeed present?

2. Separation of amino acids by HPLC

3. Separation of amino acids by partition chromatography(一)分配层析的原理： 分配系数 (Kd )

Kd = CA/CB某物质在A相中的总量

有效分配系数 (Keff):

Keff =某物质在B相中的总量(二)分配柱层析： (三)纸层析： (四)薄层层析： (五)气相色谱(气液层析，属分配色谱):

Rf = X/Y

薄层层析：

氨基酸的光学性质1. 旋光性: AA的旋光符号和 大小取决于R基的 性质,且与pH有关.

2. 紫外吸收:

Trp, λ=280nm;Tyr, λ=275nm; Phe, λ=257nm;

朗伯-比尔(Lambert-Beer)定律 在特定波长下，溶液中物质的光吸收与其浓度 C(以mmol-1为单位)和溶液中光径长l(以cm 为单位)成正比。 A=εCl

3.8 Determining the Sequence

of Amino Acid residues Proteins can be sequenced in two ways: 1. Real amino acid sequencing

2. Sequencing the correspondingDNA in the gene

Protein Sequencing Procedure in real amino acid sequencing1. Determination of polypeptide chain number; 2. If more than one polypeptide chain, separate them; 3. Cleave (reduce) disulfide bridges; 4. Determine amino acid composition of each chain; 5. Determine N- and C-terminal residues; 6. Cleave each chain into smaller fragments by sitespecific proteases or chemicals; 7. Determine the sequence of each chain by Edman degradation or MS; 8. Reconstruct the sequence of the protein from the sequences of overlapping fragments; 9. Determination position of disulfide bridges.

<1> Edman degradation

§1. Determine N- terminal residues1. FDNB:

DNP

DNP

DNP