Engelbert Buxbaum

Steady-State Kinetics

The theoretical aspects of enzyme activity, inhibition, and inactivation have been discussed in a separate volume [44]. More extensive treatments of both theory and practice of enzyme kinetics may be found in [27, 68, 69]. Ready-to use rate equations for most mechanisms may be found in the otherwise dated monography by Segel [372], rate equations may also be obtained by a computer program described in [438].

Determination of Protein Concentration

Protein Determination Using Absorbance at 280 nm Determination of protein concentration by ultraviolet absorption (260 nm to 280 nm) depends on the presence of aromatic amino acids in proteins. Tyrosine and tryptophan absorb at approximately 280 nm. Higher orders of protein structure also may absorb UV light or modify the molar absorptivities of tyrosine and tryptophan, and thus the UV detection is highly sensitive to pH and ionic strength at which measurement is taken. Many other cellular components, and particularly nucleic acids, also absorb UV light. The ratio of A280/A260 is often used as a criterion of the purity of protein or nucleic acid samples during their purifi cation. The real advantages of this method of determining protein concentration are that the sample is not destroyed and that it is very rapid. Although diff erent proteins will have diff erent amino acid compositions and thus diff erent molar absorptivities, this method can be very accurate when comparing diff erent solutions of the same protein.

Fluorescent Staining of Gels

Certain transition metal complexes show intensive fluorescence when bound to proteins. They can be used to stain gels after electrophoresis with a sensitivity approaching that of silver staining, but in a much simpler and more reproducible procedure. Stains can be prepared easily and at a fraction of the cost of commercially available reagents.Hydrophobic dyes can be used to stain gels without fixing; they do not interfere with later blotting or electro-elution.

3-D Structures

This introduction to NMR must be necessarily brief. Further information can be found in [344, 436] and references therein.

Protein Secondary Structure

There are several methods available that can be used to gain information on secondary structure, apart from X-ray crystallography, which remains the ‘gold standard’.

Testing Whether or Not a Model Fits the Data

Usually we use mathematical models to describe our data. Regression methods (linear or non-linear) are then used to determine the parameters of such models that result in the best fit, for example by the least-squares criterium. There is one problem with this approach, however: If the model is inappropriate then the fitting procedure will spit out meaningless parameters without any warning. A classical example – often seen in student’s lab reports – is the fitting of a straight line through data that form a curve.

Reagents for Other Groups

The reactivity of alcohols in aqueous solutions is low, most reagents prefer to react with more nucleophilic groups like amine or thiol.

Folding and Unfolding of Proteins

Proteins are stabilised by the difference between the bond energies between interacting amino acids and the energy of interaction between these amino acids and water (several MJ∕mol each). Protein folding decreases not only the enthalpy, but also the entropy of the molecule. Hence the total stabilisation energy is ≈ 20–60kJ∕mol which should be compared with thermal energy k T ( ≈ 2kJ∕mol at room temperature).

Spontaneous Reactions in Proteins

One would like to think of proteins as relatively stable entities, at least in the absence of modifying enzymes. Alas, this view is mistaken. As time passes, proteins can undergo spontaneous modifications that change their structure and function.

Principles of Optical Spectroscopy

Many analytical techniques involve the interaction of a sample with light (IR, visible and UV). While microwave radiation excites rotation of a molecule (ΔE ≈ 4kJ∕mol) and IR-light oscillatory movement of atoms around their position in a molecule (vibration, ΔE ≈ 40kJ∕mol), visible and UV light move electrons to higher energy levels (ΔE ≈ 400kJ∕mol). X-rays excite nucleons (ΔE ≈ 4000kJ∕mol).