Human Biochemistry Volume 1 - Murray R. 1993

Biomolecules and Biochemical Methods
General experimental approaches used in biochemistry

This approach consists of three parts: 1) isolation of Biomolecules and Organelles present in The Cell (see the previous section on subcellular fractionation); 2) Determination of the Structure of biomolecules; 3) use of various preparations to analyze the Functions of biomolecules and their METABOLISM (i.e., Synthesis and degradation processes).

1. Isolation of biomolecules. As in the case of organelles, elucidating the function of any biomolecule first requires obtaining it in a pure state. Table 2.5 lists the main Methods used for the Separation and purification of biomolecules. We will not discuss them in detail here; some of them will be briefly described in other PARTS OF THE book.

Class="center">Table 2.5. Main Methods for the separation and purification of biomolecules. Most of these methods are suitable for analyzing components present in cell extracts and other biochemical preparations. Obtaining most biomolecules in a pure form generally requires the sequential use of several methods. Details on the application of each method can be found in the relevant manuals.

Almost always, purifying biomolecules to a state of homogeneity (i.e., free from contamination by any other biomolecules) is achieved only through the sequential application of several such methods.

It is important to bear in mind that progress in biochemistry depends on The Development of new Methods of Analysis, purification, and structure determination. For example, the field of Lipid Biochemistry was truly revolutionized by the Introduction of gas-liquid and Thin-Layer Chromatography into practice. The analysis of membrane and many other Proteins was extremely difficult until the advent of Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE): the application of sodium dodecyl sulfate leads to the "solubilization" (dissolution) of A number of proteins that previously could not be brought into a soluble state. Following such solubilization, electrophoresis can then be performed. The development of DNA Cloning and sequencing methods (determining The sequence of monomers) truly revolutionized The Study of Nucleic Acids and biology in general.

2. Determination of The structure of biomolecules. Once biomolecules are purified, their structure can be determined. This is an absolute prerequisite for a successful detailed Study of the correlation between Structure and function. The main methods used for analyzing the structure of biomolecules are listed in Table 2.6. These should be familiar to readers who have studied organic chemistry. The Specificity of action of a number of Enzymes allows them to be used as powerful tools for elucidating Structural Features of certain biomolecules. Theoretical and technical progress, which has increased the resolving power of mass spectrometry and NMR spectroscopy, has contributed to the widespread use of these Methods for determining molecular structures. For example, the extremely complex structure of carbohydrate chains that make up certain biomolecules, particularly Glycoproteins, has in many cases been established using high-resolution NMR spectroscopy. The most detailed information on the structure of biomolecules is provided by X-ray crystallography methods. It is thanks to their use that the detailed structure of various Proteins and Enzymes was established, and the DNA double helix was discovered.

Table 2.6. Main methods for determining the structure of biomolecules

3. Study of the functions and metabolism of biomolecules using various preparations. Early biochemical studies of humans and animals were carried out at the whole-Organism level. Examples include the study of Respiration and The Fate of compounds entering the organism. However, it soon became clear that the whole organism is too complex to obtain clear Answers to various questions. Many problems encountered when working with the whole organism were eliminated by preparing simpler samples and studying them in vitro. Table 2.7 lists the various types of preparations currently used to study biochemical processes; most of the information contained in this book was obtained precisely using such preparations. In the table, preparations are listed in order of decreasing complexity. However, The Use of simpler preparations also has its limitations. In vitro experimental results can be erroneous, for example, when cell homogenization releases enzymes that partially degrade the compounds under study.

Table 2.7. Hierarchical sequence of preparations used to study biochemical processes

General strategy for studying biochemical processes

In this book, the main focus is on complex biochemical processes (e.g., Protein Synthesis, Muscle contraction), including Various metabolic pathways. A metabolic pathway is a set of reactions responsible for the synthesis of complex compounds from simpler ones and for the degradation of a compound into end products. A particular complex biochemical process or metabolic pathway sometimes manifests at the whole-organism level. Muscle contraction is an example of this kind. We know that glucose is a source of energy for humans and other animals, which means that in The Human Body it must be degraded (metabolized) with the release of energy. However, in order to get a complete picture of how glucose metabolism occurs in the cell — and we do not yet have such a picture (particularly regarding the regulatory mechanism) — studies must be carried out at other levels. Fig. 2.3 presents various types of observations and analyses that provide a comprehensive Overview of an entire biochemical process, such as glucose degradation and energy release (a process known as Glycolysis). This scheme is broadly applicable to all major biochemical processes discussed in this book, and thus illustrates the general strategy for studying biochemical processes; this should be kept in mind when considering any biochemical process (glycolysis, Fatty acid oxidation, etc.).

Additional remarks on preparative work and research strategy

It is useful to make a few more remarks on some of the points listed in Table 2.7 and Fig. 2.3. 1. The possibility of artifacts in no way diminishes the absolute necessity of isolating and identifying each component participating in a biochemical process in a pure state; without this, it is impossible to understand how the process proceeds at THE MOLECULAR LEVEL. This is confirmed by numerous examples that we will encounter later. 2. It is important to learn to reconstruct the process in vitro through the systematic Selection of individual components. If, after assembling all system components, the process still does not proceed, this may mean that some important component was missed during identification and was not added during assembly. 3. Thanks to recent technological advances (e.g., in NMR spectroscopy and positron emission tomography), it has become possible to detect specific biomolecules at the whole-organism level and monitor changes in their content over time. This allows the analysis of many complex biochemical processes in vivo. 4. Only after observations made at different levels yield consistent results can one speak with confidence about real progress in understanding the biochemical process under study. If significant discrepancies arise when using different approaches, the cause of these discrepancies should be sought until a rational explanation is found. 5. The combination of different research levels and various types of preparations described above can be used to identify Biochemical changes in animals associated with altered metabolism (e.g., during restricted or enhanced Nutrition) or with specific diseases (diabetes mellitus, Cancer). 6. Most of the methods and approaches described above are applicable to the study of normal and pathological human Cells and Tissues. In doing so, care must be taken to ensure that only freshly prepared samples are used, and special attention should be paid to ethical issues associated with human experimentation.

Fig. 2.3. Strategy for studying a metabolic process or metabolic pathway. The Procedure used does not necessarily have to include all the steps listed here. However, applying these very approaches generally makes it possible to elucidate the details of biochemical processes or metabolic pathways. This scheme has been used in a general form to study the Major Metabolic Pathways discussed in subsequent chapters.

Use of isotopes in biochemistry

The introduction of isotopes into biochemical practice in the 1930s played a tremendously important role. Previously, it was very difficult to label biomolecules in order to subsequently track their transformations during metabolism. In pioneering studies, primarily by Schoenheimer and his colleagues, stable isotopes (²D, ¹⁵N) were used to solve many biochemical problems, and their fate was subsequently tracked using mass spectrometry. For example, Certain Amino acids, sugars, and Fatty acids containing stable isotopes were synthesized and then added to the diet of animals or to in vitro preparations to monitor their metabolism (determine half-life, conversion into other biomolecules, etc.). It was in this way that many aspects of protein, carbohydrate, and Lipid Metabolism were elucidated. It became clear that metabolism is a highly active process: most compounds in the cell are constantly synthesized and degraded, although the rates of these processes can vary greatly. Summing up all these results allowed Schoenheimer to formulate THE CONCEPT OF the "Dynamic State of body constituents" (or dynamic nature of metabolism).

The subsequent use of radioactive isotopes and instruments capable of quantifying them was of great importance. Stable and radioactive isotopes widely used in working with biological systems are listed in Table 2.8. Their application has played a decisive role in the development of a number of areas in biochemistry. Many studies of simple and complex biomolecules in vivo and in vitro rely heavily on the use of isotopes. The recent progress achieved in nucleic acid sequencing and in the development of radioimmunoassay methods for measuring very small quantities of compounds in biological systems was also largely driven by the application of isotopes.

Table 2.8. Isotopes most widely used in biochemical research