441
OF ~IOSOAAIIKE OXIDASE DETERAIIKATION
July 196s I
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Figure 1.-Lineweaver-Bnrk plot for m-iodobenzylamine (siibstrate) and solubilized beef liver mitochondria (source of JIXO).
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FigLire S.-Lineiveaver-Burk plots obtained for two oxygen concentrations. Solubilized beef liver mitochondria are used as a sourc'e of JIAO and rn-iodobenxylamirie as a substrate. The reaction rate, Q, expresses tlie increase in optical density a t 253 mp/mg./min.
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OPTICAL DENSITY
Figure 2.--Rerording of monoamine oxidase activitv. The ciirves represent increase in optical density a t 253 mp as a function of time: A, "solubilized" molise liver homogenate (1.67 mg. of liver/ml.), run a t a roller speed of 2.54 cm./min.: B, "solitbilided" homogenate of human brain gray matter (3.33 mg./ml.), riiu a t a roller speed of 6.2 mm./miri. drawn. This solution was diluted with an equal volume of cold phosphate buffer, whereas materials obtained from tissues with lower h1.40 activit,y, e.g., brain, were used without, further dilution. Since 253 mp is not far a r a y from the protein peak, one is limited in the amount of the enzyme source which can be used in the reaction mixture. For the quantities described above, large slit, widths are required. These did not seem to interfere with the measurements (see Figure 1). 3litochondria were prepared according to Hogeboom, et a1.,14 and Cotzias and Dole,11a as described previou Choice of m-Iodobenzylamine as a Substrate.-Our studies on the occurrence of eutopic and dystopic complexes in the AIAO reaction led to the recognition that with the exception of fluorine, the introduction of any substituent in the meta position led to iiiuch better substrates than benzylamine.1bs15 By far the highest reaction rates, as expressed by maximum velocity ( V ) ,were observed with m-iodobenzylamine. This substrate was therefore -elected for our further studies. I n beef and mouse liver a value (Jf higher than that for tyramine, the classical substrate of JIAO, was found. Relationship between Oxidation and Deamination.-The use of m-iodobenzaldehyde as a basis for activity determinations rests on the assumption that the product formed by dehydrogenation of the amine does not undergo further oxidation. If the ratio of equivalents of oxygen taken up to molerules of NH3 released by a n LIAO system is greater than one ( O / S H 3 > I ) , it is an indivation t,hat the primary product, of the enzyme r c w t ion undergoes further oxidat ion. We found that, m-iodoIwnxylamine is resistant toward this kind of oxidation. Using five concentrations of t,his substrate ranging from 2 to 10 mill Lvit ti liver mitochondria of seven species, and running the incuIlation for 30 min., we observed that the O/N& was never more (14) G. H. FIogebooin, W. C . Sclineider. a n d G. E. Pallade, J . BzoZ. Chem., 172, 615) (1948). (1.5) E. A . Zeller, .'inn. S.Y . Acad. Sc?., 107, 811 (1963).
away from unity. The same was true for liver homogthan enatec;. For t,yramine we found ratios as high as 3 and 4 (cat and dog liver). Furthermore, the synthetic m-iodobenzaldehyde does not undergo any oxidation perceptible to manometric and photometric measurements under the conditions of our experimental procedure. I n this respect, m-iodobenzylamine seems to be an ideal substrate. Assay Procedure.-The standard test solutions consist of 0.2 ml. of enzyme solution and 2.8 ml. of 0.36 X 10-3 31 m-iodobenzylamine in phosphate buffer, 0.067 Af, p H i.2 (giving a final concentration of 0.33 X 10-3 AI), or 0.2 ml. of enzyme in phosphate buffer only. Vessels containing substrate and buffer solutions are shaken under oxygen a t 38" for at least 10 min. and are then sealed with Parafilm (manufactured by Marathon, American Can Co., llenosha, Kis.); a separate erlenmeyer flask is used for each sample to avoid excessive oxygen dilution after breaking the seal. The enzyme solution, however, is kept in an ice bath and is not flushed with oxygen. The change in absorbance is measured a t a wave length of 253 mp in a RIodel 14 Cary recording spectrophotometer a t a compartment temperature of 38". -4n example of actual recordings is given in Figure 2. The liver samples, 1.67 mg./ml., are run for approximately 2 min. a t a roller speed of 2.54 cni./min., while the brain sample, 3.3 mg./ml., are measured for 4 min. a t a speed of 6.2 mm./ min. Rates are expressed as differences in optical density/g. of enzyme/min. I n contrast to evaluations of manometric experiments, where oxygen uptake is calculated for the total amount of enzyme present, all photometric data reported here pertain to milligrams or grams of enzyme present in 1 ml. The Beckman ultraviolet spectrophotometer and Gilford four-channel photometer have also been found to be suitable for these measurements. Oxygen mnsumption is determined under similar conditions as photometric changes with regard to buffer, teniperature, and oxygen concentration, as described previously.llb~l~ Effect of Oxygen Concentration on the Reaction Rate.Earlier observations17 regarding the effect of oxygen concentration on M A 0 activity were confirmed by our study. As shown in Figure 3, the rate of degradation of m-iodobenzylarnine was much ilower in presence of air than of pure oxygen. .At 0.33 X l O - - 3 JI concentration, the ratio of the two rates was 0.51. For this reason, the reaction system was saturated with oxygen (see preceding paragraph).
Results Effect of Enzyme Concentration on the Keaction Rate.-& shown in Figure 4, the reaction rabe is a (16) E. A . Zeller and S. Sarkar, J . Bioi. Chcm., 237, 2333 (19623. (17) (a) F. J. Philpot, Biochem. J., 31, 856 (1937): (b) H. I. Kohn, ibid., 31, 1693 (1937).
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500L IO
F HUMAN E'igiirr .;, -~(,'lliiii):iris(iti o f pliotoriietiic. ailti in:iiic~riietrii, tleterminatioiis of inoiionmine oxitlase. For the photometric. procediire (1)l:ic-k vnlumiis) 0.33 x 10- .1/ :~iicI for the riiai~ometric? (white colnmns) 3 X IOV3 .lI ,ii-iod(iheiiz?-larniiie is used. The other c'onditioiis regarding hriffer, pH, temperatiire, :iiiti oxygeii c,oiic.ei~trat~ioiiare the same for both methods. Thc rwiilts are giveii as (.j,,xand I vdiies, indicating peqniv. of osygen/g./hr. a n d difference o p t i d densit>-/g./hr. (iiiitial vclorit ies ).
liiieai. fiiiictioii of the ciizynie quantity added in t,he foi-iii of honiogenatc or mitochondria \vit'liiii a wide range.
Effect of Substrate Concentration on the Reaction Rate. -- T o find out wliether our inetliod could b c x applied f o r liiiit6c studics.
n-tt iiir-ostigated thc effect of substratc, cwiccwtratioii oil tlic rcaction d o c i t y a i d ohtaiiicd I,inc\~ea\-er-13ui.k diagrams of the type show1 in Figurc. 1. These iiidicatc that, thc Xchaelis:\lcntclii relationship appears to be satisfied within a wide raiigci of concc>ntratioiisj s c ~also Figure 3 ) .
Comparison between Manometric and Photometric Determinations.----Theresults of the photometric and thv niaiionietric methods n.ere compared with each othvi. foi. :L numbel, of different materials. Sincar t h c disc:o\yry of this viizyiii(1by €Iarch.'E tlic. lattvr proccdui~c~ has h c c l i i c~xtc~nsi\~ely uscd in eiiz)-mological aiid pharinat i 8 ) 31.
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DETERMIXATION OF ~IOXOAXINE OXIDASE
July 1965
413
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'0 NEG. LOG. IPRONIAZID CONC. Figure 6.-Determination of pIjo (negative logarithm of concentration producing 50Yc inhibition) for iproniazid and solubilized beef liver mitochondria under standard conditions. Preincubation of enzyme with iproniazid lasted 15 min.
pargylamineZ4the degree of inhibition increases rapidly. The new photometric procedure, requiring very short reading periods, s e e m to be suited for the analysis of these fast processes and thus for the screening of 11-40 inhibitors. I n Figure 7 the inactivation of M A 0 by 2 p X pargylamine and o-chloropargylamine is denionstrated; with the latter compound, substantial inhibition was noted after 10 sec.
Discussion 1:or the assessnient of nionoaniine oxidase ( L A O ) activity there is only o m available method which is iiiore sensitive than the one described here. I n this procedure the nieasurement is carried out with the help of isotopically labeled substrates20b,cand yields discrete points of the reaction curve only. The same is true for the method based on the disappearance of serotonin2j or on the formation of indoleacetic acid.26 We are mainly interested in initial velocities, which can be deteriniiied most easily with manometric and photometric measurements. Only the latter two shall be discussed henceforth. Since the original investigations on ,\IAO,is the conventional manometric procedures have been extensively used for the study of this enzyme. They require large amounts of honiogenates, 20-200 mg./ml. or more or the equivalent amounts of mitochondria and microsomes, as compared with the 0.3-4 nig./ml. needed for the present method. This feature of the Warburg technique makes it alniost iinpossible to analyze the distribution of X 4 0 in organs of low enzymic activity obtained from sniall laboratory animals, such as niouse brain, and to avoid inactivation of inhibitors by high tissue concentrations (see section on Inhibition Reactions). With preparations of low AL40 activity a n apparent or true lag phase is often observed and erratic readings are encountered, so that not too rarely it becomes difficult to determine (241 .I. D. Taylor, A . A . Wykes, T.C . Gladish, and W . R. Rlartin, S n t u r e , 187, Y 4 1 (1960).
(25) A . Sjoerdsina, T. E. Siiiitli, T. D. Steveiison, and S. Udeiifriend, Proc. SOC.Ezptl. Bid. M e d . , 89, 36 (19.55). ( 2 6 ) W.Lovenberg, R. J. Levine, and A . Sjoerdsma, 6.Pharmacal. Ezpil. Therap.. 136, 7 (1962).
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2 3 4 5 6 7 LENGTH OF PREINCUBATION
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Figiire 7.-Inhibition of beef liver monoaniiiie oxidase by (A) pargylamine and (€3) o-chloropargylamine. Solutions of the two inhibitors ( 2 pJ1) were incubated with solubilized beef liver mitochondria for varioiis lengths of time before the addition of nz-iodobenzylamine.
the initial velocity precisely. I n addition, the oxygen uptake may not only be caused by the 3IAO reaction pel. se, but also by oxygen consumption due to other reactions with endogenous substrates. Although it is possible to avoid this extraneous oxygen consumption by adding cyanide and seniicarbazides to the measuring systeni,1ib'2ithese agents niay complicate the experimental situation considerably. As an example we mention the strong effect of cyanide on the inhibition of _\IAO by hydrazine derivatives.2ib~3aIn spite of these shortcomings, the manonietric procedure is still a very valuable tool in the investigation of MAO, in particular of substrate pattenis. ni-Iodobenzjrlaniirie appears to be a suitable substrate not only for photometric, but also for manonietric determinations. Other photometric procedures of &IO nieasureiiients are based on the oxidation of benzylaminei3 and kynuraniine.i2 That benzylamine is often not a suitable substrate has been mentioned above. Kynuramine is deaminated and the resulting aldehyde condenses intramolecularly to form 4-hydroxyquinoline. The latter compound has a much lotver molar extinction coefficient a t 360 nip than the starting product. The difference in molar optical density between the starting and end product is approximately 5000, as compared with 8400 for the pair, m-iodobenzylamine and In-iodobenzaldehyde. The initial optical readings with 0.1 i d 1 kynurainine are relatively high, so that small differences in optical density may be of doubtful value. One could avoid this difficulty in part by following the increase in optical density between 310 and 335 nip which accompanies the forniation of 4hydroxyquinoline. But even in this case, the molar extinction coefficient of the substrate ranges from 1000 to 3000. No experimental data for this procedure, however, are given. The sensitivity of the two methods niay be best compared by indicating the differences in molar optical density induced by the same material. For the rat, the values are 2.5 (Figure 4 in ref. 12) and 16 (Table I1 of this paper). (27) (a) 1%. Illascliho, D lliclitei, and 11 d l l o s s r i i a l i n , U i u ~ I t ( ? t i J , 31, 2187 (19377, (b) X H Greasy, zbid.. 64, 178 (1956) (28) T. E Smith, H. Weissbach, and S. Udenfriend, B z o c h e m z s l r y , 2, i 4 6 (1963).
