On an equal weight basis polymyxin B and EM 49 which do not contain tyrosine or tryptophan yielded the same colour intensity as proteins in the Folin-Lowry and biuret methods. But, in the absence of reagent C (alkaline copper reagent) polymyxin B and EM 49 yielded no colour in the Folin-Lowry method. Mono-, di- and tri-formyl polymyxins B formed identical amounts of coloured complexes as polymyxin B in the two methods. However, the tetra- and penta-formyl polymyxins B yielded only one-fifth and one-sixth, respectively, of the expected colour in the Folin-Lowry method. Similarly, 40% and 30%, respectively, of the anticipated amount of colour is formed in the biuret method. Formylated and methylated lysozyme and bovine serum albumins form only 70–75% of the expected colour in the Folin-Lowry method. Since formation of colour by reduction of Folin reagent, in the Folin-Lowry method, is at least partly due to complexes of copper, it was inferred that polymyxin B as well as its mono-, di- and tri-formyl derivatives on the one hand and the tetra- and penta-formyl derivatives on the other differ in their ability to complex Cu(II) The former group of compounds was indeed found to complex as many as three Cu(II) ions whereas the tetra- and penta-formyl polymyxins B complexed only one equivalent, under conditions of excess Cu(II). Under conditions of low Cu(II), polymyxin B and all its derivatives complexed only one Cu(II). In proteins, sites other than amino groups which complex Cu(II) probably play a major role in the reduction of the Folin reagent, since methylated lysozyme and bovine serum albumin yield 70–75% of the colour formed by the unmodified proteins in the Folin-Lowry reaction.

An inorganic pyrophosphatase has been purified to apparent homogeniety from Indian cobra(Naja naja) venom, with a ten-fold increase in specific activity. The enzyme activity is intrinsic to a protein fraction in the venom which is normally termed cardiotoxin, cobramine, cytotoxin and so on. The enzyme shows a lowK_m (70 μI) and high heat stability. The enzyme was active against sodium pyrophosphate; it also hydrolyses a few mononucletides and sugar phosphates at much lower rates. The physiological significance of inorganic pyrophosphatase in venom is discussed.

Cardiotoxin II of the Indian cobra(Naja naja) contains approximately four Mg²⁺ per mol. Complete demetallation of the toxin is achieved by three cycles of treatment with ethylenediamine tetraacetate and gel filtration. Reconstitution of toxin by treatment of the apo-protein with Mg²⁺ restores metal content and inorganic pyrophosphatase activity only to the extent of two atoms/mol and 65%, respectively. Use of Mg (II)-EDTA in the reconstitution experiment yields restoration of half the original enzyme activity. Mg²⁺ is required for the inorganic pyrophosphatase action of the toxin. A definitive statement on the non-essentiality of Mg²⁺ for the lethal toxicity of the toxin is not possible at present, although experimental observations indicate that demetallated toxin is as toxic as the native toxin. Based on this and the differing sensitivities of the enzyme and toxic activities of the toxin to heat, it is suggested that the reaction centres in the toxin for the two activities are different and that the pyrophosphatase activity is not causally connected with the lethal toxicity of the toxin

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4.0), 0.01 N H₂SO₄ (pH 2.0) and Tris-HCl buffer (pH 8.5) on oxidation of tyrosine residues was exa mined. In comparison with 0.01 N H₂S O₄, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodo-succinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3.75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.