Vol. 56 ORIENTATION OF GROUPS IN PROTEIN MONOLAYERS 513 Lysyl Bulky 2. At pH 13 the un-ionized c-NH, groups ofthe residue hydrocarbon lysine residues tend to enter the non-aqueous phase, oil oil air roup Peptide \ group especially at the air-water interface when the chain z z Water neighbouring amino acid residues in the peptide EvQ_OI O chain are favourable. 3. The 'unavailability' ofsuch e-NH2 groups at water water ork Glutamyl this interface and in the native protein residue may be of O/W:. low and O/W: very high A/W: moderate major biological importance. mnoderate film pressures film pressures film pressures The author is deeply indebted to Prof. Sir Eric Rideal, (1-6 m.'I mg:-') (< 1-0 M., mg.-) (1-6 m.1 mg.-') F.R.S., for his continued encouragement throughout the (a) (b) course of this work. Fig. 4. Orientations of the -(CH2)4NH2 chains in proteins at (a) the oil-water interface, and (b) the air-water 1,EFERENCES interface. Alexander, A. E. & Teorell, T. (1939). Tranm. Faraday Soc. in 35, 727. the aqueous phase either A/W monolayers at Cohn, E. J. & Edsall, J. T. (1943). Protein8, Amino-acids and moderate pressures or in the native protein (where Peptide8, pp. 358-60. New York: Reinhold. the van der Waals forces of cohesion between the Davies, J. T. (1951 a). Nature, Lond., 167, 193; Z. Elektro- hydrocarbon side chains are also very powerful). chem. 55, 559. When the protein is unfolded at a lipid-water Davies, J. T. (1951 b). Proc. Roy. Soc. A, 208, 224. interface the e NH2 is much more readily available Davies, J. T. (1953). Tran8. Faraday Soc. 49, 949. (Fig. 4), and in this we may find some explanation of Fraenkel-Conrat, J. & Fraenkel-Conrat, H. (1950). Biochim. the curious and specific biological phenomena biophy8. Acta, 5, 89. mentioned in the introduction. Gerovich, M. & Frumkin, A. (1936). J. chem. Phy8. 4, 624. Haurowitz, F. (1950). Chemi8try and Biology of Proteins. SUMMARY New York: Academic Press. Haurowitz, F. & Schwerin, P. (1940). Enzymologia, 9, 193. 1. The potentials of monolayers of various Holtfreter, J. (1946). J. exp. Zool. 101, 355. proteins and amino acid polymers, including a new Needham, L. (1951). A8pect8 of Form, ed. by L. L. Whyte. synthetic amphoteric amino acid polymer, have London: Humphries. been measured at the interfaces air-water and Olcott, H. S. & Fraenkel-Conrat, H. (1947). Chem. Rev. 41, oil-water, at pH's 2, 6-8 and 13. With this new 151. Porter, R. R. (1948). Biochim. biophy8. Acta, 2, 105. polymer (poly-1: 1:2:-L-lysyl-L-glutamyl-L-leucine) Reid, E. (1951). Nature, Lond., 168, 955. all the phenomena shown by protein fihls can Sanger, F. (1952). Advanc. Protein Chem. 7, 1. now be reproduced. Other polymers studied Stallberg, S. & Teorell, T. (1939). Trans. Faraday Soc. 35, were poly-L-lysine, poly-DL-leucine, poly-1:1-y- 1413. methyl-L-glutamyl-DL-phenylalanine and poly-1:1- Tristram, G. R. (1949). Advanc. Protein Chem. 5, 83 DL-lysyl-L-glutamic acid. (Table 37). A Colorimetric Method for the Determination of the Principal Metabolites of Nicotinic Acid in Human Urine BY W. I. M. HOLMAN Medical Re8earch Council Department of Experimental Medicine, Univer8ity of Cambridge (Received 17 July 1953) Recent work has shown that N-methyl-2-pyridone- are not normally present (Reddi & Kodicek, 1953). 5-carbonamide and N-methylnicotinamide are the It therefore seems likely that the sum ofthe urinary main metabolites of nicotinic acid normally ex- excretions of N-methyl-2-pyridone-5-carbonamide creted in human urine (Holman & Lange, 1949; and N-methylnicotinamide, expressed as nicotinic 1950), that no more than a trace of nicotinamide is acid, may prove to be a reliable index of nicotinic excreted (Reddi & Kodicek, 1953), and that nico- acid status in man. Existing methods for the deter- tinic acid, N-methylnicotinuric acid betaine and mination of these metabolites, however, are pyridine nucleotides, and probably nicotinuric acid, laborious and time consuming and, since the Biochem. 1954, 56 33 5;14 W. I. M. HOLMAN I954 methods involve quite different techniques, it is technique of Bratton & Marshall (1939), as modified by difficult for one analyst to determine both sub. Smith, Finkelstein, Aliminosa, Crawford & Graber (1945). stances at the same time. A colour was not always formed, however, particularly when It has also been shown (Holnan & Lous, 1951) the amount of hypobromite was large in relation to that of that N-methyl-2-pyridone-5-carbonamide tends to the pyridone. The amount of bromine utilized during the reaction with the pyridone, as estimated by thiosulphate distribute itselfin the total body water ofman. It is titration, increased with the excess ofhypobromite added up possible that, if a simple method were available for to as much as 8 g. moles Br/mole of pyridone. Since only its estimation, this pyridone might provide the 0 5 g. mole Br/mole is required for the Hofmann reaction basis for a method of estimating body water. (e.g. see Sidgwick, Taylor & Baker, 1937), these results The present investigation was made in an attempt suggested that side reactions occurred, probably leading to to devise a rapid colorimetric method for the simul- extensive oxidative breakdown of the pyridone molecule. taneous determination of N-methyl-2-pyridone-5- Side reactions could probably have been largely pre- carbonamide and N-methylnicotinamide, based on vented by avoiding an exce#s of bromine, but this technique the conversion of the carbonamide group into an is not likely to be suitable for analytical purposes, particu- larly in the presence of urine, which contains many sub- amino group by treatment with hypobromite, and stances capable of reacting with hypobromite. An alter- diazotization and coupling of the amino compound native possibility was examined, namely, adding an excess formed to yield an azo colour. The hypobromite ofhypobromite at room temperature to ensure combination reaction (Hofmann reaction) has long been used in with the carbonamide group of the pyridone, and then preparative chemistry, but was not thought to removing the excess before heating the solution. Of various have been adapted for analytical purposes. Although substances tested as agents for the removal of excess of there was a possibility that urine might contain bromine, phenol was the most satisfactory, enabling substances capable of giving azo colours by the intense colours to be obtained even when a large excess of diazotization process alone, it seemed unlikely that hypobromite had previously been added to the pyridone. Under suitable conditions a colour could be formed in the any urinary constituent other than derivatives of presence ofas much as 5000 g. moles Br/mole pyridone. By nicotinic acid would require both hypobromite the use of phenol, azo colours could also be produced from treatment and diazotization for the development of nicotinamide, N-methylnicotinamide and N-methyl-2- an azo colour, since this series ofreactions is specific pyridone-3-carbonamide. In every case the amount of test for aromatic and heterocyclic amides. substance required to give a colour of suitable intensity for After the present work had been completed, it was colorimetric measurement was 1-50,ug. The chief optical discovered that Goodyear & Murphy (1944) had properties of the colours formed are summarized in Table 1. proposed a method based on the Hofmann reaction A trace of brownish yellow colour was produced by the for the estimation ofnicotinamide in pharmaceutical reagents in the absence ofmetabolites ofnicotinic acid. This colour increased in intensity during the first 45 min. after preparations. They did not, however, apply their addition of the coupling agent, but altered only slightly method to other metabolites ofnicotinic acid nor to thereafter. Its absorption at 590 mit. was equivalent to the analysis of biological materials. The relatively about 0 4,ug. of N-methyl-2-pyridone-5-carbonamide, and large amount (75-325 pg.) of nicotinamide required that at 500 mp. to about 3ug. of N-methylnicotinamide for a determination was an important factor limiting chloride. Coenzyme I (diphosphopyridine nucleotide) did the application of the method. not yield a colour, the trace of orange produced by a pre- paration of this substance being accounted for by the nicotinamide which was present as an impurity. EXPERIMENTAL Optimum conditions of heating and concentrations of Preliminary experiments showed that a purple azo colour NaOH, bromine and phenol were ascertained. Nicotin- could be formed from N-methyl-2-pyridone-5-carbonamide amide and N-methylnicotinamide required for maximal by heating with hypobromite solution, and then acidifying colour production a slightly more alkaline hypobromite the solution and diazotizing and coupling according to the solution than the pyridones. It was also found with each of Table 1. Properties of azo colours produced Light absorption at various wavelengths* Colour Time to reach Substance produced maximal intensity 500 mp. 570 mp. 580 mit. 590 mP. Nicotinamide Orange 8 min. (after 30 min. 100 1-2 0.5 fades 4%/hr.) N-methylnicotinamide Orange 8 min. (after 30 min. 100 - 2-7 0*3 fades 4%/hr.) N-methyl-2-pyridone-5- Purple 1 hr. (stable 1-6 hr. then 45 100 98 92-5 carbonamide fades 0-1 %/hr.) N-methyl-2-pyridone-3- Blue 2-5 hr. (stable 2-5-21 hr.) 27 98 100 98 carbonamide * As percentage of maximal value. VoI. 56 DETERMINATION OF NICOTINIC ACID METABOLITES 515 the four substances that colour production could be con- methyl-2-pyridone-3-carbonamide, was transferred to a siderably increased by protecting the solution from light Pyrex, rimmed, 6 x I in. test tube and diluted to 4 ml. with during the reaction.