It has been a tremendous honour being asked by Keith Wilson, John Walker and Cambridge University Press to take on the role of editorship for this eighth edition of Principles and Techniques. In designing the content, we extend the long and successful tradition of this text to introduce relevant methodologies in the biochemical sciences by uniquely integrating the theories and practices that drive the fields of biology, biotechnology and medicine. Methodologies have improved tremendously over the past ten years and new strategies and protocols that once were just applied by a few pioneering laboratories are now applied routinely, accompanied by ever more fluent boundaries between core disciplines – leading to what is now called the life sciences.

In this eighth edition, all core methodologies covered in the previous edition have been kept and appropriately updated and consolidated. New chapters have been added to address the requirements of today's students and scientists, who operate in a much broader area than did the typical biochemists one or two decades ago. The contents of this text are thus structured into six areas, all of which play pivotal roles in current research: basic principles, biochemistry and molecular biology, biophysical methods, information technology, -omics methods and chemical biology. Of course, due to space restraints that help to keep this text accessible, the addition of new material required us to consolidate and carefully select content to be kept from the previous edition. These decisions have been guided by the over-arching theme of this text, namely to present principles and techniques. Importantly, our experience as teachers has always been that many undergraduates are challenged by quantitative calculations based on these principles, and hence we have continued the tradition of this text by including relevant mathematical and numerical tools as well as examples.

Indeed, new chapters on data processing, visualisation and Python have example applications that can be accessed from the CUP website to aid understanding.

Sadly, two authors of previous editions have passed away: John Fyffe died shortly after the seventh edition was launched and Alastair Aitken passed away in 2014. Also, Keith Wilson, John Walker and Robert Burns decided to retire, and we thus invited several new authors to contribute to this eighth edition.

FROM BIOCHEMISTRY AND MOLECULAR BIOLOGY TO THE LIFE SCIENCES

Biochemistry is a discipline in the natural sciences that is chiefly concerned with the chemical processes that take place in living organisms. Starting in the 1950s, a new stream evolved from traditional biochemistry, which, until then, mainly investigated bulk behaviour and macroscopic phenomena. This new stream focussed on the molecular basis of biological processes and since it put the biologically important molecules into the spotlight, the term molecular biology was coined. Importantly, molecular biology goes beyond the mere characterisation of molecules. It includes the study of interactions between biologically relevant molecules with the clear goal to reveal insights into functions and processes, such as replication, transcription and translation of genetic material.

Major technological and methodological advances made during the 1980s enabled the development and establishment of several specialised areas in the life sciences. These include structural biology (in particular the determination of three-dimensional structures), genetics (for example DNA sequencing) and proteomics. The refinement and improvement of methodologies, as well as the development of more efficient software (in line with more powerful computing resources), contributed substantially to specialist techniques becoming more accessible to researchers in neighbouring disciplines. What once had been the task of a highly specialised scientist who had been extensively trained in that particular area has consistently been transformed into a routinely applied methodology. These tendencies have pushed the feasibility of cross-disciplinary studies into an entirely new realm, and in many contemporary laboratories and research groups, methods originally at home in different basic disciplines are frequently used next to each other.

It is thus not surprising, that in the past 15–20 years, the term ‘life sciences’ has been used to describe the general nature of studies and research areas of a scientist working on studies related to living organisms.

THE EDUCATION OF LIFE SCIENTISTS

The life sciences embrace different fields of the natural and health sciences, all of which involve the study of living organisms, from microorganisms, plants and animals to human beings. Importantly, even satellite areas that are methodologically rooted outside natural or health sciences, for example bioethics, have become a part of the life sciences.

INTRODUCTION

Spectroscopic techniques are based on the interaction of light with matter and probe certain features of a sample to learn about its consistency or structure. Light is electromagnetic radiation, a phenomenon exhibiting different energies. When light interacts with matter, it can emerge with other characteristics and properties, and therefore different molecular features of matter can be probed. Conceptually, matter can either annihilate (absorb) or create (emit) light (discussed in this chapter), or it can scatter light (discussed in the next chapter, Section 14.3.5). An understanding of the properties of electromagnetic radiation and its interaction with matter leads to an appreciation of the variety of types of spectrum and different spectroscopic techniques, and their applications to the solution of biological problems.

In this section, we introduce the basic principles of interaction of electromagnetic radiation with matter and then examine absorption and emission of light by matter and the corresponding molecular spectroscopic techniques of absorption (Sections 13.2, 13.3, 13.4) or emission (Sections 13.5, 13.6). Atomic spectroscopy (Section 13.7) is based on the same principles, but uses light of higher energy.

The applications considered use visible or UV light to probe consistency and conformational structure of biological molecules. Usually, these methods are the first analytical procedures used by a biochemical scientist, and are routinely employed in a variety of experimental approaches (e.g. enzyme kinetics, Section 13.8).

Properties of Electromagnetic Radiation

Electromagnetic radiation (Figure 13.1) is composed of an electric (E) and a perpendicular magnetic vector (M), each one oscillating in plane at right angles to the direction of propagation, resulting in a sine-like waveform of each, the E and the M vectors. The wavelength ƛ is the spatial distance between two consecutive peaks (one cycle) in the sinusoidal waveform and is measured in submultiples of metre, usually in nanometres (nm). The maximum length of the vector is called the amplitude. The frequency ν of the electromagnetic radiation is the number of oscillations made by the wave within the time frame of 1 s. It therefore has the units of 1 s −1 = 1 Hz. The frequency is related to the wavelength via the speed of light in vacuo, c = 2.998 × 10 8 m s −1 by

DEFINITION AND CLASSIFICATION OF ENZYMES

Catalytic reactions in biological processes are facilitated by two types of catalysts, enzymes and ribozymes. Whereas enzymes are proteins, ribozymes consist of ribonucleic acid (RNA). Most enzymes are much larger than the substrates they process, but this is not a requirement (for example, restriction enzymes that cleave DNA). The catalytic features of enzymes arise through a particular three-dimensional arrangement of functional groups in a small number of amino acids (see Section 23.4.2) in the active site of the enzyme. The geometrical arrangement of such groups enables productive interactions with the bound substrate and leads to formation of a transition state for which the energy barrier (activation energy) is significantly reduced as compared to the non-catalysed reaction (see Figure 23.1). Consequently, the reaction rate is increased by several orders of magnitude relative to the non-catalysed reaction. Importantly, enzymes do not alter the position of equilibrium of the reversible reactions they catalyse, rather they accelerate establishment of the position of equilibrium for the reaction.

Many enzymes are the key players in metabolic or signalling pathways. In these coordinated pathways, enzymes are collectively responsible for maintaining the metabolic needs of cells under varying physiological conditions (Section 23.5). Through their individual catalytic activities, they control the rate of a particular metabolic or signalling pathway. A range of regulatory mechanisms operates to allow short-, medium- and long-term changes in activity (Section 23.5.1). Therefore, the over- or under-expression of an enzyme can lead to cell dysfunction, which may manifest itself as a particular disease. Hence, enzymes have been the most important target in the development of therapeutics, and typical drugs for the treatment of pathological conditions act as inhibitors of particular enzymes (Sections 23.5.3). Other clinically relevant applications include monitoring of enzyme levels in assessment of disease states. For example, damage to the heart muscle as a result of oxygen deprivation following a heart attack results in the release of cellular enzymes into extra-cellular fluids and eventually into the blood. Such release can be monitored to aid diagnosis of the organ damage and to make a prognosis for the patient's future recovery (Section 10.3).