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Fifth international symposium on affinity chromatography and biological recognition
IX
trendsin analyticalchemistry,vol. 2, no. 11, 1983
Affinity chromatography A report from the Fifth International Symposium on Affinity Chromatography and Biological Recognition, held in Annapolis, MD, USA from 13 to 17 June 1983. This symposium covered a wide range of topics in a particularly rapidly moving area of analytical chemistry. Receptors, protein-protein interactions, DNA-protein interactions and biospecific ligands are highlighted in this report. The receptor field was reviewed by Pedro Cuatrecasas, who highlighted the three most significant advances: the way in which receptors work in pairs, their relationship with receptosomes, and the subtle use of pH differentials in vesicles. Receptor density and the different conformational changes brought about by antagonists and agonists show how agonists have a vital cross-linking role in activating receptors. Calmodulin is a tine example of a multi-functional protein where the protein-calmodulin interaction can be neatly altered by calcium. Not only does this confirm our ideas on receptors but it also provides a series of useful affinity chromatography applications (lectures by Klee and Myohinen). In fact, calmodulin has emerged as one of the most interesting multifunctional proteins, and the mechanism of calcium mediated protein-calmodulin interactions neatly fits into the receptor study. More complex protein-protein interactions can be studied using allosteric proteins. Haemoglobin variants provide a valuable model for such regulatory systems (Ackers). The amount of excellent information which could be obtained from restricting the selection of haemoglobins to non-lethal variants was impressive. Far more dramatic changes in haemoglobin sub-unit interactions could presumably be made by genetic engineering, but thoughtful use of the 400 or so ‘live’ mutations have made possible a thermodynamic map of the energy changes which describe haemoglobin dimer-tetramer equilibria during partial oxygen saturation. At this sym0165-9936/83/$01.,X,
posium there were two lectures on haemoglobin-based systems. Recent advances in the use of immobilized phenylboronic acids in the assay of glycosylated haemoglobins were discussed (Dean). Unfortunately, nobody dared ask how multi-site partial glycosylation affects the complex energy changes in haemoglobin allostery described above. Simpson’s lecture demonstrated how some elegantly simple DNA-protein interactions rule our lives; the way in which DNA polymerase recognizes certain parts of DNA helix was admirably demonstrated in 20 min of slides depicting space-filled DNA models. Contact points are easily picked out by these models. The polymerase model suggests a ubiquitous pair of helices which are needed to align the protein into the major groove of the DNA. With this model one can explain the mode of interaction of DNA with restriction enzymes, repressors, polymerases, etc. The whole lecture reminded this author of the period a decade ago, when Rossmann proposed that other widespread structural unit - namely the nucleotide binding site. Simpson also showed that the eukaryote story requires an explanation of how nucleosomes are phased and packaged. How two enzymes are held down on a surface and made to talk to each other was one of the reviews we have come to expect at these symposia. Using bifunctional reagents, more efficient immobilized enzyme reactors can be built. Holding multi-subunit enzymes down by one subunit provides an excellent opportunity to fish for enzymes by subunit exchange since most subunit-subunit interactions can be dissociated fairly gently. Chiancona described how to do this for a wide variety of enzymes. The technique is still in its infancy but is growing in applications. Stellwagen addressed himself to the eternal problem of ligand concentration. Is this parameter negotiable? Interesting preliminary experiments suggest that some insight into the improvement of column capacities, i.e. ligand efficiency, may be obtained by developing this work. Antibody speci-
ficity stimulated a wide-ranging debate. Several speakers presented work designed to challenge antibodies with widely different antigens (Inman) or partial protein sequences (Anflnsen). Hydrophobicity plays an important part in our understanding of protein-ligand interactions (Shaltiel). Using the latter to obtain the best chromatographic separations requires careful choice of the hydrophobic term in combination with the best ligand (Yon). The effects of hysteretic adsorption and multi-site interactions explain many anomalies in the current practice of affinity chromatography (Jennissen). The use of biospecific ligands (especially metals and substrates) to modulate the adsorption and desorption of proteins from both conventional and affinity gels is becoming increasingly sophisticated and now even includes metal chelate chromatography (Porath). The use of immobilized monoclonal antibodies is widely recognized as a gentler way of recovering high value, low volume proteins. Whilst bovine growth hormone does not come into this category, there are many proteins which do. The isolation of proteins from low Ka antibody columns can be achieved with relatively mild desorbing agents such as 0.75 M sati. (Carlton). Meir Wilchek invariably provides a stimulating lecture. He provided two at this conference; the first was on the latest developments in the design of Erlich-type magic bullets and the second on hazard-free cyanogen bromide activations - a subject in which he is an undoubted master. Other activation methods, such as using carbonyldiimidazole (Hearn) and dipsylation (Scouten) were discussed in depth. P. D. G. DEAN P. D. G. Dean is Reader in Biochmnistry, The University, Liverpool, UK.
TrAC LIBRARY
EDITION
With the Trend? in Analytical Chemistry
Library Edition you receive normal monthly issues plus an annual compendium ofthe archival contents, with an author and subject index, reprinted on high quality paper and bound in hard back. See the Subscription Card for details. @ 1983
Elrevicr Science Publishers
B.V.
trendsin analyticalchemistry,vol. 2, no. 11, 1983
Affinity chromatography A report from the Fifth International Symposium on Affinity Chromatography and Biological Recognition, held in Annapolis, MD, USA from 13 to 17 June 1983. This symposium covered a wide range of topics in a particularly rapidly moving area of analytical chemistry. Receptors, protein-protein interactions, DNA-protein interactions and biospecific ligands are highlighted in this report. The receptor field was reviewed by Pedro Cuatrecasas, who highlighted the three most significant advances: the way in which receptors work in pairs, their relationship with receptosomes, and the subtle use of pH differentials in vesicles. Receptor density and the different conformational changes brought about by antagonists and agonists show how agonists have a vital cross-linking role in activating receptors. Calmodulin is a tine example of a multi-functional protein where the protein-calmodulin interaction can be neatly altered by calcium. Not only does this confirm our ideas on receptors but it also provides a series of useful affinity chromatography applications (lectures by Klee and Myohinen). In fact, calmodulin has emerged as one of the most interesting multifunctional proteins, and the mechanism of calcium mediated protein-calmodulin interactions neatly fits into the receptor study. More complex protein-protein interactions can be studied using allosteric proteins. Haemoglobin variants provide a valuable model for such regulatory systems (Ackers). The amount of excellent information which could be obtained from restricting the selection of haemoglobins to non-lethal variants was impressive. Far more dramatic changes in haemoglobin sub-unit interactions could presumably be made by genetic engineering, but thoughtful use of the 400 or so ‘live’ mutations have made possible a thermodynamic map of the energy changes which describe haemoglobin dimer-tetramer equilibria during partial oxygen saturation. At this sym0165-9936/83/$01.,X,
posium there were two lectures on haemoglobin-based systems. Recent advances in the use of immobilized phenylboronic acids in the assay of glycosylated haemoglobins were discussed (Dean). Unfortunately, nobody dared ask how multi-site partial glycosylation affects the complex energy changes in haemoglobin allostery described above. Simpson’s lecture demonstrated how some elegantly simple DNA-protein interactions rule our lives; the way in which DNA polymerase recognizes certain parts of DNA helix was admirably demonstrated in 20 min of slides depicting space-filled DNA models. Contact points are easily picked out by these models. The polymerase model suggests a ubiquitous pair of helices which are needed to align the protein into the major groove of the DNA. With this model one can explain the mode of interaction of DNA with restriction enzymes, repressors, polymerases, etc. The whole lecture reminded this author of the period a decade ago, when Rossmann proposed that other widespread structural unit - namely the nucleotide binding site. Simpson also showed that the eukaryote story requires an explanation of how nucleosomes are phased and packaged. How two enzymes are held down on a surface and made to talk to each other was one of the reviews we have come to expect at these symposia. Using bifunctional reagents, more efficient immobilized enzyme reactors can be built. Holding multi-subunit enzymes down by one subunit provides an excellent opportunity to fish for enzymes by subunit exchange since most subunit-subunit interactions can be dissociated fairly gently. Chiancona described how to do this for a wide variety of enzymes. The technique is still in its infancy but is growing in applications. Stellwagen addressed himself to the eternal problem of ligand concentration. Is this parameter negotiable? Interesting preliminary experiments suggest that some insight into the improvement of column capacities, i.e. ligand efficiency, may be obtained by developing this work. Antibody speci-
ficity stimulated a wide-ranging debate. Several speakers presented work designed to challenge antibodies with widely different antigens (Inman) or partial protein sequences (Anflnsen). Hydrophobicity plays an important part in our understanding of protein-ligand interactions (Shaltiel). Using the latter to obtain the best chromatographic separations requires careful choice of the hydrophobic term in combination with the best ligand (Yon). The effects of hysteretic adsorption and multi-site interactions explain many anomalies in the current practice of affinity chromatography (Jennissen). The use of biospecific ligands (especially metals and substrates) to modulate the adsorption and desorption of proteins from both conventional and affinity gels is becoming increasingly sophisticated and now even includes metal chelate chromatography (Porath). The use of immobilized monoclonal antibodies is widely recognized as a gentler way of recovering high value, low volume proteins. Whilst bovine growth hormone does not come into this category, there are many proteins which do. The isolation of proteins from low Ka antibody columns can be achieved with relatively mild desorbing agents such as 0.75 M sati. (Carlton). Meir Wilchek invariably provides a stimulating lecture. He provided two at this conference; the first was on the latest developments in the design of Erlich-type magic bullets and the second on hazard-free cyanogen bromide activations - a subject in which he is an undoubted master. Other activation methods, such as using carbonyldiimidazole (Hearn) and dipsylation (Scouten) were discussed in depth. P. D. G. DEAN P. D. G. Dean is Reader in Biochmnistry, The University, Liverpool, UK.
TrAC LIBRARY
EDITION
With the Trend? in Analytical Chemistry
Library Edition you receive normal monthly issues plus an annual compendium ofthe archival contents, with an author and subject index, reprinted on high quality paper and bound in hard back. See the Subscription Card for details. @ 1983
Elrevicr Science Publishers
B.V.