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Dlvlnyisulphone-activated agarose to which mercaptoethanol IS coupled showed very setectlve group adsorption of human serum protems, m particular the ~mmunogIobul~ns The adsorptxon Increases markedly m the presence of high concentrations of neutral water-structure formmg salts and IS dlstmct from adsorptions based on hydrophobic mteractlon A characterlstlc feature of this new type of adsorbent 1s the structure of the groups attached to the polymer, @, I e , R-S-CH,-CH,-SO,-CH,-cH,-O-Q, where R IS a small ahphatlc residue Our results mdlcate that the thloether sulphur and the adJacent sulphone group act cooperatlvely and are apparently necessary to mamtam the dlstmct behavlour of such dbsorbents

Several prototypes of aromatic (Ar) and non-aromatic (NoAr) cation-exchange ligands suitable for capture of proteins from high conductivity (ca. 30 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal cation-exchangers were found by screening a diverse library of multi-modal ligands and selecting cation-exchangers resulting in elution of test proteins at high ionic-strength. Candidates were then tested with respect to breakthrough capacity of bovine serum albumin (BSA), human IgG and lysozyme in buffers adjusted to a high conductivity. By applying a salt-step or a pH-step the recoveries were also tested. We have found that aromatic multi-modal cation-exchanger ligands based on carboxylic acids seem to be optimal for the capture of proteins at high-salt conditions. Experimental evidence on the importance of the relative position of the aromatic group in order to improve the breakthrough capacity at high-salt conditions has been found. It was also found that an amide group on the alpha-carbon was essential for capture of proteins at high-salt conditions. Compared to a strong cation-exchanger such as SP Sepharose Fast Flow the best new multi-modal weak cation-exchangers have breakthrough capacities of BSA, human IgG and lysozyme that are 10-30 times higher at high-salt conditions. The new multi-modal cation-exchangers can also be used at normal cation-exchange conditions and with either a salt-step or a pH-step (to pH-values where the proteins are negatively charged) to accomplish elution of proteins. In addition, the functional performance of the new cation-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 10 days. For BSA it was also possible to design cation-exchangers based on non-aromatic carboxyl acid ligands with high capacities at high-salt conditions. A common feature of these ligands is that they contain hydrogen acceptor groups close to the carboxylic group. Furthermore, it was also possible to obtain high breakthrough capacities for lysozyme and BSA of a strong cation-exchanger (SP Sepharose Fast Flow) if phenyl groups were attached to the beads. Varying the ligand ratio (SP/Phenyl) could be used for optimizing the function of mixed-ligand ion-exchange media.

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50 % of the isolated inhibitors and were further purified to a homogeneous state.The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 "C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HC1. The intact inhibitors were destroyed when the peas were cooked at 100 "C or when they were toasted at 130 "C.The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20 % of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).Proteins which inhibit the activity of proteolytic enzymes have been isolated from the tissues of a variety of animal and plant species. Their distribution, properties and biological significance are presented in recent general reviews [ 1,2] and proceedings of research conferences [3,4]. While proteinase inhibitors in most leguminous seeds have been extensively studied, those from Cicer arietinum (commonly called chick peas, bengal gram, garbanzo or coffee pea) have so far not been purified and characterized, although Borchers and Ackerson [5] reported in 1947 that these seeds contain a high concentration of trypsin inhibitor. Their results were confirmed by Sohonie and Bhandarkar [6] who also showed that the crude inhibitor preparation was acid stable and fairly thermostable. Later, Abramova and Chernikov [7] showed that extracts from chick peas inhibited trypsin and chymotrypsin but not pepsin.Since chick peas constitute an important source of dietary protein in some parts of the world, and in view of the limited investigation carried out on their inhibitors, we have undertaken to purify and study them in detail. Such studies can contribute, at least in part, to a understanding of their significance in animal nutrition. In this paper the purification of the inhibitors including some of their physical and chemical properties is described. The...

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