Biochemistry in the Lab

A Manual for Undergraduates

Benjamin F. Lasseter

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9780429957376
• Edition: 1
• Topic: Medicina
• Subtopic: Bioquímica en medicina

Most lab manuals assume a high level of knowledge among biochemistry students, as well as a large amount of experience combining knowledge from separate scientific disciplines. Biochemistry in the Lab: A Manual for Undergraduates expects little more than basic chemistry. It explains procedures clearly, as well as giving a clear explanation of the theoretical reason for those steps.

Key Features:

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• Presents a comprehensive approach to modern biochemistry laboratory teaching, together with a complete experimental experience

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• Includes chemical biology as its foundation, teaching readers experimental methods specific to the field

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• Provides instructor experiments that are easy to prepare and execute, at comparatively low cost

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• Supersedes existing, older texts with information that is adjusted to modern experimental biochemistry

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• Is written by an expert in the field

This textbook presents a foundational approach to modern biochemistry laboratory teaching together with a complete experimental experience, from protein purification and characterization to advanced analytical techniques. It has modules to help instructors present the techniques used in a time critical manner, as well as several modules to study protein chemistry, including gel techniques, enzymology, crystal growth, unfolding studies, and fluorescence. It proceeds from the simplest and most important techniques to the most difficult and specialized ones. It offers instructors experiments that are easy to prepare and execute, at comparatively low cost.

1 Buffers

A large conceptual difference that exists between the way biochemists think and the way organic chemists think has to do with solvents and buffers. The organic chemist will need his reactions to be in an environment of a certain polarity, either a protic environment or an aprotic one, with extreme purity, and at temperatures that range from freezing cold up to hundreds of degrees. Thus, organic chemists agonize over which solvent to use and how to prevent any other chemical contaminating their solvent and how to get a proton to react in a non-polar environment. The biochemist on the other hand, has a much happier and more relaxed lab condition. There is only one solvent for proteins or nucleic acids: water. If you need a source of protons, the 55 M water will always be an excellent source. So far from avoiding contaminating chemicals, the biochemist will deliberately seek them out in order to control the pH of this watery environment. These contaminants, from the organic chemist’s point of view, are the buffers the biochemist uses.

A buffer is a chemical system that resists changing pH from a certain value. It is merely a weak acid (HA) and its corresponding weak base (A⁻), existing both together in the same environment. The acid component can prevent pH from increasing due to added hydroxide (OH⁻) as shown in the following chemical reaction:

$$\text{HA}(aq)+{\text{OH}}^{-}(aq)\to {\text{H}}_2\text{O}(\text{l})+{\text{A}}^{-}(aq)$$

(1.1)

The pH does not increase in this case because there is no OH⁻ (aq) left over. Similarly, the base component can prevent pH from decreasing due to added protons (H₃O⁺ in water) as shown in the following chemical reaction:

$${\text{A}}^{-}(aq)+{\text{H}}_3{\text{O}}^{+}(aq)\to {\text{H}}_2\text{O(l)}+\text{HA}(aq)$$

(1.2)

As before, the pH does not change, this time because there is no H₃O⁺ (aq) left over. Thus, the pH does not shift very far from a certain value, so long as there are significant quantities of both the acid and base component present. Practically speaking, this resistance to pH change occurs most significantly when the concentration of the acid is no more or no less than ten times the concentration of the base.

The buffer environment is very important to the biochemist because it determines the charge state of the biomolecules. In general, the concentration of protein molecules or nucleic acid molecules will be much less than the concentration of any buffering component. Therefore, they do not cause the pH to become a certain value as much as they become a certain value due to their own pKa values on any ionizable groups. If the pH is less than the pKa, any ionizable side chain will be in its protonated acidic form, with whatever consequences for charge that has. The reverse is true if pH is more than pKa.

Consider: if a protein has some aspartate residues (pKa ≈ 4), some lysine residues (pKa ≈ 12) and some histidine residues (pKa ≈ 6), if it is in an environment where pH = 10, then every one...