1203
COMMUNICATIONS TO THE EDITOR
reductionY are inefficient and give only small yields. I n a typical preparation, 50 ml. of a solution 0.6 AVin hydrochloric acid of tin(I1) chloride containing 0.43 mmole of tin were introduced into a reaction flask connected to a series of traps suitable for the collection of the condensable products. The reaction system was swept with nitrogen, and the nitrogen stream was maintained throughout the reduction. From a dropping tube, 30 ml. of a 5yc aqueous sodium borohydride solution were added over a period of 20 minutes. -%tthis point the reduction was considered complete. The produced gases were passed through a trap held a t -23' to remove most of the water vapor and through a trap kept a t -196' to collect the stannane. The crude product was purified by fractionation through a trap maintained at - 112'. The yield of stannane was 0.39 mmole or 84yb based on the amount of tin taken. The product was identified by its vapor pressure a t -105" (39 mm.) and a t -78' (19s mm.), values which are in agreement with those reported by Paneth.4 The identification was verified by permitting 0 . i 3 mmole of stannane to decompose into the elements. After seven days the deconiTiosition was judged complete and 1 . E mmoles of hydrogen were found compared to 1.30 mmoles expected for stannane. It has been observed that the yield of the hydride is dependent upon the acid concentration, the effect of which is still being investigated, and upon the concentration of tin. This latter effect is illustrated by the following data : 11g. of tin per ml. of s o h .
11
6 1 3 1
Yield of stannane,
C/o
9 18 25 3i
red blood cell preparations which we have previously demonstrated are capable of synthesizing protoporphyrin in vitro. We have found that not only can homogenized duck erythrocyte preparations synthesize protoporphyrin from &aminolevulinic acid but t h a t a soluble cell-free extract oE the duck red blood cell can effect the synthesis. I t can be seen from Table I, that the supernatant fluid obtained on high speed centrifugation (12 X l o 3 to 100 X l o 3g) is almost as active as the homogenate and t h a t even a lyophilized preparation of the cell-free extract still retains its synthetic activity. On the other hand, although the whole red blood cell or a gently hemolyzed preparation ol duck erythrocytes can synthesize protoporphyrin from glycine and succinate,3-5 the ability to convert these substrates t o protoporphyrin is lost on disruption of the structure (Table I ) . It would appear that on homogenization the functional activity of only those enzymes that are involved in the condensation of succinate with glycine is lost. TABLE I HEMINS Y N T I I E S I Z E I ) ~-AMINOI,EVULISIC AkCID-5-C'4( [ J . ~ ~ M C . l % f % l). A S l ) ISIC A c I D - ~ ' - C (i~ l , O~, j MC. < a h I . ) I N DIFFERENT Rei) BLOODCELLPREPARATIOYS"
C O M P A R I S O S OF
C" Act,. v i t v in
hemiti sample. Expt.
1
2
3
B y an analogous reaction small but sigriificant yields of bismuthine have been obtained. The preparation of these hydrides and those of related elements is under continuing study.
DEPARTMENT OF CHEMISTRY
-4CTIVITIES OF
FROM
84
( 3 ) F. Paneth a n d E. Rabinonitsch, Be?., 67B,1877 (1!12$1. (4) F. P a n e t h , 1 x 7 , Haken a n d It. Kabinowsitsch, ibid., 67B, 1808 (1924).
Vol. 76
Substrate, acid
Succinic (0.05 mLZ.jb Succinic (0.03 m M ,jb 6-Aminolen1linic (0.013 m l f . ) 8-Aminolevulinic (0.013 mh'l.) 6-Aminolevulinic (0.009 mh1. j 8-Aminolevulinic (0.009 m h l . i 6-Aminolevulinic (0.009 m M . ) 8-Ainiriolevuliiiic (0,009 m M i
-1
Red cell preparatioii
c.11.m
:"
Hemolyzed
-
I-Iomogenizecl
i
Hemolyzed
4300
Homogenized
4300
Homogenized
2ZNI
Supernatant
( 12
l(i00
Supernatant (17 X I O 3 g) Supernatalit ( 1011
1 (i00
x
1 0 3 gi
15'JO
X I(Pq1
8-iliriiriole\uliiiic SripcrtiatailL (12 (iI.O~J9mL1. 1 x 1O"l 6-.~1ni11olcl-uli11ic 1,yophilizecl prqiii.
2000
I !3 10 GEORGE\V. SCIIAEFFEK ( i l . o ~ l o1nnr. 1 SISTER M. EMILIUS, 0.S.I; Each preparation reprcseiitcd 25 inl. of duck blood a ~ ~ t l RECEIVED J A A U A R Y 15, 1954 Plus 0.38 mhf. of prepared as previously described.".' iion-radioactive glyciiie.
ST.LOLXSUXIVERSITY ST. LOUIS,MISSOURI
THE SYNTHESIS OF PROTOPORPHYRIN FROM 6AMINOLEVULINIC ACID IN A CELL-FREE EXTRACT'
\Ve have recently reported that 6-aininolevulinic acid can replace the two substrates, "active" succinate and glycine for porphyrin synthesis.' I n order to investigate porphyrin synthesis in greater detail we have begun t o fractionate duck
Further proof that 6-aininolevulitiic acid is indeed the precursor for porphyrin synthesis was obtained by degrading a hemin sample synthesized froin 6-arninolevulinic a ~ i d - , X ' ~ . .The " ~ ~6-carbon atom of the latter coiiipound should label the S ~ I carbon atoms of protoporphyrin as those which we have previously found arise from the a-carboii atom of glycine6 since the latter carbon atom is the
(1) This work was supported b y grants from the National Institutes oi Health, United States Public Health Service (RG-l128(C5)), from the American Cancer Society o n the recommendation of t h e Committee on Growth uf t h e S a t i o n a l Research Council, a n d from t h e Rockefeller Foundation. ( 2 j I). Shemin a n d C . S . Russell, THISJ O U R N A L , 76, 4873 (1953).
( 3 ) D. Shemin, I. h f . London a n d D. Kittenberp, J. B i d . C h e w , 173,799 (1948); 183, 757 (1950). ( 4 j D . Shemin a n d S. Kumin, i b i d . , 1 9 6 , 827 (19521. ( . j j I. If.London a n d 11.Yamasaki, Feiiernlioii Pt'oc., 11,250 (IU521 (6) J . Wittenberg a n d D . Shemin, J . Riol. Cham., 186, 103 (19XJ). (i) L).Shemin a n d J. Wittenberg, ibid., 192, 315 (1921).
Sir:
V
COMMUNICATIONS TO THE EDITOR
Feb. 20, 1954
source of the &carbon atom of the 8-aminolevulinic acids2 It can be seen from Table I1 t h a t the same CI4 distribution pattern was found in protoporphyrin synthesized from 8-aminolevulinic a ~ i d - 5 - C ' ~ and from glycine-2-C14; 50Y0 of the C 1 4 activity resides in the pyrrole rings and SOY0 in the methene bridges.
30
1205
38 42 46 50 Mass number, m / e . mass spectrum resulting from hydrolysis of pentaborane.
34
TABLE I1 Fig. 1.-Residual DISTRIBUTIOX OF Cl4 ACTIVITY IN PROTOPORPHYRIN SYNTHESIZED FROM &AMINOLEVULINIC ACID-5-cl4 AND FROM GLYCISE-2-C" was very small. The amount of hydrogen generMolar activity (Yo)in,fragments ated in the hydrolytic reaction increased with time. of porphyrin synthesized from 6-Aminolevulinic Glycine-2.~2'4 acid-c-C'4, % (ref. G ) , 70
Mass spectra taken immediately and a t short intervals after mixing pentaborane and water vapor Protoporphyrin 100 100 showed no residual pattern except for hydrogen. Pyrrole rings A B 23.5 24.6 The residual pattern given above is very similar (methylethylma1eimide)n to the mass spectrum of tetraborane3 for which the Pyrrole rings C + D principal monoisotopic species is B4H6. We be25.2 25.3 (Hematinic acid)" lieve the first step in the hydrolysis of pentaborane PyrroleringsA + B + C + D 49.7 49 , 9 to be the removal of a borine by the "bound Methene bridge carbon atoms 50.3 .50 . 1 water" with subsequent generation of hydrogen. l a Obtained from protoporphyrin as previously de~cribed.~,' From a comparison of the models for pentaborane4 Our finding that 8-aniinolevulinic acid is an inter- and tetraborane5 the removal of any one of the four mediate in porphyrin synthesis has been confirmed basal borons of pentaborane would leave the tetraborane skeleton. in two recent c o m m u n i ~ a t i o n s . ~ ~ ~ T h a t the "bound water" in silica removes a (8) A. Neubergerand J. J. Scott, Naluve, 171, 1093 (1953). borine from pentaborane can also be demonstrated (9) E. I. R. Dresel a n d J. E. Falk, ibid., 171, 1185 (1953). by boron and hydrogen balances of what is retained DEPARTMENT OF BIOCHEMISTRY on the silica and what is recovered in the volatile COLLEGEOF PHYSICIANS AND SURGEONS DAVIDSHEMIN COLUMBIA UNIVERSITY TESSA ABRAMSKY portion of the products. By adding water to the SEW YORK32, N. Y. CHARLOTTE S. RUSSELL silica after all volatile material has been pumped off the solid, one can measure the amount of active RECEIVED JANUARY 22, 1954 hydrogen gasometrically and the amount of boron by titration with mannitol and standard base. FIRST STEP I N THE HYDROLYSIS OF PENTABORANE Silica must be filtered from the solution before Sir : titration of the boron. I n one experiment pentaNormally, the identification of intermediate borane from a carbon tetrachloride slush ( - 23') products resulting from the hydrolysis of boron was passed through a bed of silica a t room temhydrides is difficult to obtain because of the ease perature into a liquid nitrogen trap. The nonwith which they hydrolyze; however, by immobiliz- condensable gas (hydrogen) was compressed into a ing the water molecules we were able to obtain gasometer by means of a toepler pump. The evidence for the occurrence of such intermediates amount of hydrogen gas generated during the in the case of diborane.'I2 Now, by means of the pentaborane hydrolysis was approximately the mass spectrometer and with the technique of using same as that found after the addition of water to "bound water" in silica gel' we have succeeded in the silica. The ratio of active hydrogen to boron identifying the first step in hydrolysis pentaborane. in the silica was found to be slightly greater than Purified pentaborane gas was passed through a unity. Thus the over-all ratio of hydrogen (hyshallow bed of silica gel containing only bound dride) to boron accounted was ca. 2.3 instead of the water, and then led directly into a Consolidated expected value of 3.0 for borine. This difference Engineering model 21-103 mass spectrometer. can be attributed to errors of measurement because Mass spectra of the gaseous products were taken of the small amounts of material involved. The immediately and after intervals of 20, 40 and BO material caught in the liquid nitrogen trap was minutes exposure time of pentaborane to the silica found to be a mixture of pentaborane and an ungel. By stripping the mass spectrum for pure stable product which decomposed a t room temperapentaborane from these spectra, we obtained a ture over a period of hours to give hydrogen and a residual pattern whose peak heights as a function fibrous-looking solid boron hydride. of mass numbers (wz/e) is illustrated in Fig. 1. CHEMISTRY DIVISION The height of the residual peaks based upon a U. S. SAVAL ORDNANCE TESTSTATIOX I. SHAPIRO' H. G. WEISS" CALIFORNIA relative peak height of 100 a t m / e = 59 (highest PASADENA, RECEIVED JANUARY 22, 1Y54 peak height in pentaborane spectrum) was largest in the spectrum obtained after the 20 minutes (3) To be published. exposure time, and decreased with time. The (4) K. Hedberg, M . E. Jones and V. Schomaker, Puoc. A ' d A c d . residual pattern in the spectrum taken immediately Sci., 38, 679 (1952). ) M. E. Jones, K. Hedberg, a n d V. Schomaker, THIS JOURNAI after the pentaborane was exposed to the silica gel 75,( 54116 (1963); C. E. Nordman and W. N. Lipscomb, i b i d . , 76, 41lii Fragments of porphyrin
+
(1) I. Shapiro a n d H. G. Weiss. J . P h y s . C h e n z . , 5 7 , 219 (1953).
(2) H. G. Weiss a n d I. Shapiro, THISJ O U R S A L . 75, 1221 (1953)
(1053). (6) Research Dept., Mathieson Chemical Corp., Pasadena, Calif.
