COMMUNICATIONS TO THE
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under conditions adaptable to large-scale production. connective tissue. This had led to the general asOne example is the quantitative process BjHll sumption t h a t these cells are the site of synthesis 4- 3H?O 2Hr B(OH)3 BdH1~I. This repre- of heparin although the possibility has never been sents an efficient synthesis of BaHlo because BjHll is excluded t h a t they serve only a storage or excretory efficiently made from B2H6.? A h o t h e rexample is function. The a1-ailability of mouse mast cell the reaction of [(CH3)&]2BH with BjH11, convert- tumors,l 2 to 2 . 3 g. subcutaneous masses of essening nearly half of the latter to B5H9 and 3.ciCi to tially homogeneous tissue, made possible a study B6Hla. This represents the best known way to of the metabolism of heparin in mast cells, in s'itvo. obtain the hitherto very rare hexaborane, the Slices of tumor were incubated with either C"yield of which probably can be improved further. glucose or S"j-sulfate and heparin of relatively The conversion of B5Hll to BsHs was not neces- high puritJ- isolated. sarily direct, for B4Hlo also was formed, and wc TaBLE I have found t h a t [(CH3)2S]:iBH converts a s much ;ipprositnatel~-$5 g . o f tissue slices of iniiu5e i i i i i \ t rcll of a BaH10 sample to BjHs. These reac- tuiiior were incubated in Krebs--Ringer pliosplinte I)uffcr uggest interesting new ideas about the with either C'4-glucosc (3.3 ~ h I , / m l , O.3G , p C j p M . ) or Sadt e plI,/tnl., 3 p C j p h 1 . ) at 37' in :in attriosI)liere of mechanisms of borane interconversions by thc ~ ~ l f a(1.5 0 2 . T h e tissue \viis then homogenized, boiled, cooled, classical gas-phase processes. adjusted to pH 8.,5 and incubated with 25 mg. of pancreatin I n the experiments described all volatile prod- overnight, The samples were t h e n dialyzed for 18 hour. ucts were identified by their known physical prop- against running tap water, t h e insoluble material erties and hydrolytic analyses. Sumbers pre- removed h y centrifugation and sufficient S a C l added to thc supernatant solutiolis t u make them 1 .ll. Hepariii ceding formulas represent millimole quantities. precipitated b y the addition of an cycess of cetyltriTetraborane from Pentaborane-1 1.--Kearly pure j-lairitnoniuui bromide; t h e complex was then redi5BjHll (0.509 mmole) was hydrolyzed during one solved in 4 M S a C l and re-precipitated by dilutiou t o 1 A l / , n-as repeated three tirn Finally, t h e hcpariti cornminute a t 0", yielding 0.401 BIHio and O..iO(i \vas washed TT-ell with I tcr, dissolved ill 2 -11 S a C l B(OH)a; and 0.017 BjHg impurity was recovered. the quaternary a ~ n i n er loved a5 t h e iiisolublc thioXgain, the hydrolysis of 0.392 BjHll gave 0.360 late rait. The ,solution of heparin \vas thoroughly R4HI0, 0.767 H?, and 0.366 R(OH)3, with recovery zed agaiiirt distilled water before counting. Iiepariii of 0.023 BjH9. Xlloming for the BbHcIimpurity! as determined by metachromatic and carbazole aisaj-s commercial heparin (Upjohn, 120 units/Ing.) as :i the yields of B4Hlo were 99.8 and 97.(i?,Hs, proxirnatdy 70: i, of tlic n.eight vas accciuntctl for h y an impurity formerly difficult to estimate. hepurin. l l , 18, d87 ( l $ l : i J I ) r . I l t ~ n nh:q. k i n d l y ])to\-,ilc(ithe tiimiir t i i i n c , iiicrl i f ! t l l r i ? r \ t w r i ,I ' 1
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March 20, 1958
COMMUNICATIONS TO THE EDITOR
conditions while heparin is largely resistant." After hydrolysis in 6 N HC1 and chromatography according to Stoffyn and Jeanloz,s a radioactive arabinose spot (derived from glucosamine) was present. ( i )Hydrolysis of the S3j-labeled heparin in 2 N HC1 a t 100" for 1 hour removed 95Yc, and hydrolysis in 0.04 12; HC1 at 100@for 2.5 hours 4iyG of the original radioactivity indicating approximately equal incorporation of the labeled sulfate into amide and ester groups6
lC521
globin (termed FI# was isolated by chrornatography5 on the ion exchange resin IRC-50 with Developer No. 4. DNP-Globin from FII gave these results :
1 4 24
(4) J. E. Jorpes and S. Gardell, J . Biol. C h e m . , 176, 267 (1010). ( 5 ) P. J. Stoffpn a n d R . W. Jeanloz, Avch. Biochenz. niad B i o g h r s . , 63, 373 (1954). ( 6 ) J. E . Jorpei, H. Bostrom a n d V. M u t t , J . Bioi. C h c i i a . , 183, 607 ( l Y 50). LABORATORY O F CELLULAR PHVSICJLOGY AXD h'fETABOLISM
D s-'1
DNPGlycine
Valine
2.04 1.45 0.27 0 .33
n 34 0.68 1.58 1.65
D SPVal-leu
S u m of I ) K P-valine a n d nxpVal-leu
1.62 1.06:
1.96 1.74
0.12
1.77
If we assume that DNP-glycine is released within the first few minutes of hydrolysis and t h a t its destruction is by a pseudo first-order reaction, a very approximate reaction rate constant is 0.0s hr.--I and the quantity a t zero time is 2.013 residues. NATIOSALHEARTIXSTITUTE EDWARD U . KORX Thus, the main fetal component contains ai1 NATIOXAL INSTITUTES O F H E A L T H equal number (probably 2) of glycyl and valyl NBETHESDA, MARYLASD terminal residues. I t is of interest. t h a t the N RECEIVED JASUARY 31, 1958 terminal sequence Val-leu- is present in both adult _ _ _ ~ ~ human and fetal hemoglobin. Perhaps the two N-TERMINAL RESIDUES OF HUMAN FETAL hemoglobins have two identical chains with this KHEMOGLOBIN terminal sequence and differ in two other chains Sir: which have N-terminal glycine in fetal heinoglobin Porter and Sangerl and Masri and Singer2 have and the N-terminal sequence val-his-leu in normal found, respectively, 2.6 and 2 N-terminal valyl adult hemoglobin.6 residues in human fetal hemoglobin. We wish t o This investigation was supported in part by a report t h a t N-terminal glycyl residues are also pres- research grant (RG-4276(C2)) from the National ent. Institutes of Health, Public Health Service. After red cells from umbilical cord blood of (6) H. S.Rhinesmith, W. A. Schroeder and N. M a r t i n , THISJ o r r X N A L , white infants had been washed with saline and in press. hemolyzed, the hemoglobin was dinitrophenylated CONTRIBUTION S o . 2300 W , A . SCIIROEDER OF CHEMISTRY in aqueous solution3 and the heme was then rc- GATESA N D CRELLISLABORATORIES INSTITUTE OF TECHNOLOGY moved.3 IVhen this DSP-globin was hydrolyzed CALIFORXIA CALIFORNIA G E S J I MATSUI)A for one hour in refluxiiig (iN hydrochloric acid, PASADENA, RECEIVED JASUARY 9, 1038 1.12 N-terminal residues of DNP-glycine per molecule, 0.2%of DNP-valine, and 1.61 of DXP-val-leu T H E STRUCTURE O F CHAKSINE, were isolated from the ether extract of the hydrolA MONOTERPENE ALKALOID ysate. The quantities were 0.25 residues of DNP-glycine and 2.12 of DNP-valine after 24 hr. Sir: The alkaloid chaksine isolated froin the seeds of hydrolysis. These compounds were isolated and identified by procedures previously d e ~ c r i b e d . ~of Cassia absus Linn by Siddiqui and Ahmadl has The calculations of the N-terminal residues per been the subject of niany- studies, in the course of molecule assume that fetal and adult hemoglobin which it has been assigned various functional have essentially equal molecular weights, and that, group systems and structures (cj'. ref. 2). \Ye now wish t o report evidence, which together as does adult DXP-globin, 0.1 g. of air-dried fetal DNP-globin contains 1.14 kmoles of DNP-protein. with previously reported data, permits the assignThe above results from pooled clotted cord blood ment of structure I t o chaksine iodide. (Found: were substantiated by examination of a second C, 36.60; H, 5.87; h-,11.62; 0, 11.23; I, 35.19. sample of pooled clotted blood and a sample of in- Calculated for CllH2002N31.0.5H 2 0: C , 36.47; dividual unclotted blood. A difference lay in the H , 5.85; N, 11.60; 0, 11.05; I, 33.04; infrared DNP-glycine, which was 1.28 and 1.44 residues, (KBr pellet) 1720, Ifi70, 1600, 1572 cm.-l; PK;, respectively, in one-hr. hydrolysates. The differ- = 11). Chaksine has no N-alkyl and 0-alkyl group, a i i t l ence is a reflection of the variation in the amounts of adult hemoglobin and of other components that gives a negative iodoform test. A Kuhn-Koth are present and can be detected by chromatog- determination on the free base gave ;i value correrap1iy.j Consequently, to obtain more definite sporiding to one C-alkyl group. Hydrolysis of chaksine with 2 :V sodiuin hyclroxresults, the main coinponetit of cord blood heinoide gave a low yield of the ureido-hydroxy acid (1) R . R . Porter a n d 1'. Sanger. Hiorhrm. J . , 4 2 , 287 (1948). ( 2 ) .\.I. S. Masri a n d K. Singer, Avch. Biochcm. B i o p h y s . , 68, 111 11, CllH2,,N204, m.p. 122-123@. (Found: C , 53.88; (1955). H, 8.27; N, 11.45; 0, 26.27. Calculated: C, ( 3 ) H. S. Rhinesmith, \V. A. Schroeder and I,. Pauling, THIS 54.07; H, S.25; X, 11.47; 0, 26.20). The inJOURNAL, 79, 609 (1957). frared spectrum of the oily ester of I1 (CC14) (4) H. M. Jope and J. R. P. O'Hrien, "Haemoglobin." edited by F. J. W. Roughtrtn and J. C. Rendrew, Butterworths Scientific Pubshowed bands at 1740 an.-' (ester) :tnd 1710 cm.-l lications, I.ondon. 1919, 2159; J. F. Taylor and R. I.. Swarm, 1 ; ~ d (five-membered cyclic urea), P Y i J C , 8 , 259 ( 1 9 4 9 ) . ( A ) n R' Allrn IT' 1 : Srll!litvlrl i l r d 1 I i a l ~ 'I~ HIS ~ . I O I ' R X Ai T n , 1 1 ) S. Siddiqtii and Z AhnixL l u , / .li,r,/ i'ri, 2 , 121 { l < i : L i ) , 17.
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