Biomedical subjects
Publications and source records attributed to B S Baines.
2-(2'-Pyridylmercapto)mercuri-4-nitrophenol was synthesized and evaluated as a thiol-labelling reagent containing a chromophoric leaving group and as a reactivity probe by studies on its reactions with 2-mercaptoethanol and with papain (EC 3.4.22.2).
A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.
A simple spectrophotometric method for the detection of chymopapains A and B, contaminants in some preparations of papain (EC 3.422.2), was devised. If the amount of rapidly reacting thiol in the preparation, as determined by increase in A343 consequent on reaction with the two-protonic-state thiol titrant, 2,2' -dipyridl disulphide, is greater at PH8-9 than at pH4, contamination by the chymopapains is in dicated.
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4-Chloro-7-nitrobenzofurazan possesses at least one highly electrophilic centre (C-6) that is much more reactive towards nucleophiles than position C-4, the irreversible alkylating site of this reagent. Possible consequences of the electrophilic character of 4-chloro-7-nitrobenzofurazan for its application as a protein-labelling reagent are discussed.