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Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

1. A diagonal electrophoretic technique for studying the amino acid sequence around cysteine and cystine residues in proteins is described. The residues are first converted into S-aminoethylcysteine, and the protein is then treated with S-ethyl trifluorothioacetate, which trifluoroacetylates all the protein amino groups. The modified protein is digested enzymically and the resulting peptides are separated by paper electrophoresis. After exposure of the peptides on the paper to ammonia vapour, the electrophoresis is repeated, this time at right angles to the original direction. Peptides from which a trifluoroacetyl group is removed by the ammonia treatment will vacate the 45° diagonal formed by all other unaffected peptides owing to the exposure of an additional amino group and consequent increased electrophoretic mobility towards the cathode. Peptides containing lysine or S-aminoethylcysteine are readily purified by this technique. 2. The successful application of the technique to bovine insulin is described. 3. Various methods for distinguishing peptides containing lysine from those containing S-aminoethylcysteine in more complicated proteins are suggested and discussed.

Dimensionless apparent ionization constants of charged low-molecular-weight species may be obtained from paper-electrophoretic data at 20-25 degrees C with buffers (I0.1-0.5) of measured pH (1.5-12.5) containing oxalate ions. Relative mobilities rather than absolute mobilities were measured by using glycerol and m-nitrobenzenesulphonate respectively as standards of zero and unit mobility. Application of the procedure to ionizations of adenine, adenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, 3′:5′-cyclic AMP, ADP, ADP-glucose-agrocin 84 and ATP is described.

The electrophoretic behaviour of oligodeoxynucleotides on ion-exchange papers was studied systematically with particular attention to applications in sequence analysis. Complex mixtures of larger oligonucleotides were fractionated by electrophoresis on cellulose acetate followed by t.l.c. on polyethyleneimine-cellulose. The behaviour of the oligonucleotides on polyethyleneimine-cellulose and on the two-dimensional system can provide a useful guide to their sequence. Detailed results from some of these experiments have been deposited as Supplementary Publication SUP 50031 (13 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.