89

Features Section: Problem-Based Learning

Editor: C A Smith, the Manchester Metropoli tan University, U K

The Problems Page is fortunate in having two contri- butions in this issue of Biochemical Education. One is from Professor John Walker of the newly-formed Univer- sity of Hertfordshire, the other from Dr E James Milner- White of the University of Glasgow. Both sets of questions concentrate on the structures and activities of biological molecules and are mainly, but not exclusively, about proteins.

Proteins are the most diverse of biological molecules, i-3 Thus the amount of time and sheer hard work devoted in Biochemistry to devising methods for their detection and estimation, their purification and the analyses of their structures and biological functions is hardly surprising. Naturally, students of Biochemistry and, indeed Biology in general, need a basic knowledge of these topics and also an appreciation of the working practises involved in the associated experimental methods and techniques.

The use of problem-solving exercises lends itself readily to the teaching of methods for estimating quantities of proteins and investigating protein structures and functions, and these topics are the subject of some interesting questions from Dr Walker.

Interactions between molecules forms the basis of most, if not all, Biology. This has been cogently summarised by Doolittle 3 who describes how biological phenomena arise from the conformational changes in macromolecules which occur following the reversible binding between two biomolecules (typically a macromolecule and ligand). The strength of binding depends in turn upon the concen- trations of the participating molecular species and the magnitude of the association constant describing their binding. Dr Milner-White has supplied some interesting problems on this topic which cover the interactions between small organic molecules (nucleotides) and the cation Mg 2÷, and the association between a macro- molecule (an enzyme) and ligand (substrate), and finally a question illustrating some basic features of enzyme activity.

References ~ Most (all?) of the major undergraduate textbooks in biochemistry, eg Stryer, Lehninger

2Various authors (1985) Sci Amer 253(4), 34-157 3Doolittle, R F (1985) Sci Amer 253(4), 74-83

Proteins - - Calculations and Interpretative Questions

JOHN M WALKER

Division of Biosciences, School of Natural Sciences, University of Hertfordshire, Hatfield, Hertfordshire ALIO 9AB, UK

Introduction The following exercises have been frequently used in tutorial classes with students attending HNC/HND Science (Applied Biology) courses or those in the first or second year of our four-year 'sandwich' Applied Biology degree. The questions are mainly used to reinforce lectures that cover the subjects of protein purification and analysis.

Detection and estimation of proteins Proteins in solution can be detected by their absorbance at either 280 or 220 nm. At 280 nm, the conjugated rings of tryptophan and tyrosine absorb; at 220 nm the carbonyl group of the peptide bond absorbs and therefore in this case one is essentially measuring the number of peptides bonds present (some amino acid side chains make a small contribution to the absorbance at 220 nm). Question l(a) shows how important it is to understand the difference between these.

An alternative approach is colorimetry where an aliquot of the protein solution is mixed with a reagent that reacts to produce a colour. A commonly used method is the Bradford method: Coomassie Blue G250 (hma x 470 nm) binds to the protein in solution (predominantly to arginine residues but also partly to aromatic residues) to give a coloured complex (hmax 595 nm). The increase in absorb- ance at 595 nm is therefore a measure of the total amount of protein present. Question l(b) relates to this method.

Perhaps the most accurate way to determine protein concentration is to carry out an amino acid analysis. Methods for amino acid analysis are now highly sensitive and question l(c) reinforces this point.

Qu l(a) Figure la shows an ion-exchange column chro- matography experiment monitored by absorbance at 280 nm. Two proteins, X and Y, were eluted and appear to be present in approximately equal amounts (the peaks are of about equal size). Figure lb shows the same elution monitored at 220 nm: there now appears to be about five times as much protein in X as in Y.

Account for the apparently different relative amounts of X and ¥ in the two profiles. In terms of measuring the relative amounts of protein, which method is likely to be more accurate? Fig lb shows an additional peak, Z, not present in Fig la. Amino acid analysis showed Z to be a polypeptide. Suggest why this peak appears in one trace but not in the other.

B I O C H E M I C A L EDUCATION 21(2) 1993