Laboratories of Enzymology and Chemistry of Proteins

Recommended Prerequisites

General Chemistry, Biochemistry I, Biomolecular Spectroscopy (simultaneous students’ attendance).

Teaching Methods

This curricular unit consists of 7 introduction modules of theoretical-practical classes and laboratorial classes and a project to be intensively developed in two weeks. For the introduction modules, students are provided with summary information of the theme, the technology to be used and 2 or 3 key-words to be used in an internet research that must be carried out before the class. The project consists of the elaboration and autonomous implementation of a protocol for the purification of an enzyme that will be drawn from a previously presented list. Students will perform two presentations and discussions, one where the protocol will be proposed and other after the elaboration of the protocol.

Learning Outcomes

The main objective of this curricular unit is the acquisition of technical and practical knowledge of extraction, purification and characterization of enzymes.

Students are expected to acquire essential knowledge of the diversity of enzymatic activities and the physical-chemical characterization of biomolecules.

Students are also expected to apply the acquired knowledge in the design and assessment of the processes of proteins’ purification and in the characterization of enzymatic activity.

Work Placement(s)

No

Syllabus

1. Research of enzymatic activity in plant or animal raw extracts.

1.1. Enzyme assay design for different classes of enzymes.

2. Exploration of biomolecules’ physical-chemical properties in the process of separation.

2.1. Molecular Exclusion Chromatography: principles and applications.

2.1.2. Application for desalting, contrast with dialysis.

2.1.3. Protein localization in a chromatographic process.

2.2. Ion Exchange Chromatography.

2.2.1. Principles. Anionic and cationic exchangers.

2.2.2. Application: for example: Partial purification of Lysozyme.

2.2.3. Localization of enzymes in a chromatographic process.

2.3. Affinity Chromatography.

2.3.1. Principles. Affinity, pseudo-affinity.

2.3.2. Application: for example: Partial purification of LDH by pseudo-affinity chromatography.

2.4. Hydrophobic Interaction Chromatography.

2.4.1. Principles and applications.

2.4.2. FPLC and HPLC.

2.4.3. Use of HPLC in the separation and analysis of aminoacids and in the sequencing of peptides and proteins.

2.5. Electrophoresis.

2.5.1. Native electrophoresis and electrophorese in the presence of dissociating substances.

2.5.2. Two-dimensional electrophoresis.

2.5.3. Applications.

2.6. Electrotransference, immunodetection and immunoprecipitation.

2.6.1. Principles and applications.

3. Characterization and enzymatic activity.

3.1. Determination of kinetic parameters.

3.2. Activation and Inhibition.

4. Design and implementation of enzyme purification protocols.

4.1. Enzyme assay design.

4.2. Assessment of efficiency and costs of the purification protocol.

Head Lecturer(s)

Paula Cristina Veríssimo Pires

Assessment Methods

Continuous
Project: 25.0%
Laboratory work or Field work: 25.0%
Presentation and discussion of experimental results: 25.0%
Exam: 25.0%

Bibliography

SCOPS, Robert (1994). Protein Purifiction: principles and practice. 3 rd ed. New York: Springer - Verlag.