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
DNPGlycine
D s-'1 Valine
D SPVal-leu
S u m of I ) K P-valine a n d nxpVal-leu
2.04 1.45 0.27 0 .33
n 34 0.68 1.58 1.65
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 HEALTH 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 GESJI 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.
pi--.
12)
S, K.
C t i h u a n d 1 . N. R n p . .I. I n d C / Z P , ) \,,
