768
H. KAHLER, B. J. LLOYD,JR., AND M. EDEN
Vol. 56
ELECTRON MICROSCOPIC AND OTHER STUDIES ON A COPPER-CYSTINE COMPLEX Bs H. KAHLER, B. J. LLOYD, JR.,AND M . EDEN National Cancer Institute,' Bethesda, Maryland Received October $0, 1961
Cystine added to formvar coated copper screens produces clumps of fine fibers. Similar fibers were obtained when cupric salts were added to a cystine solution and placed oon formvar coated nickel screens. The fibers form upon drying and do not exist as such in solution. Their diameter is 85 A. Various other amino acids were tried with copper, and various metal salts were mixed with cystine, but none gave a similar fiber formation. Two H + ions are released for each copper-cystine complex formed, the ratio of copper to cystine in the complex being unity. The fibers were seen when the solutions varied in pH from 3 to 8.8. The complex in solution consists of small units which aggregate on vigorous blending into large fibers. * The use of copper screens in the electron microscopic study of biological material which may contain cystine gives rise to the possibility of fibrous artifacts.
The fibrous state is of wide occurrence in biology, cystine solution. Since the use of coppcr in the reduced (metal) with cystine probably involves a set of osidaa few familiar examples being denatured proteins, state tion-reduction reactions,il the use of cupric salts with collagen, cellulose, fibrinogen-thrombin clots and cystminewould be a chemically simpler system, free from nucleic acid. The present report is concerned with oxidation complications. Solutions of cupric chloride, nitrate, sulfate or acetate fibers obtained from a complex of cystine and copaddcd to the cystine solution and dried on formvar per. There appears to be no reference to such fi- when coated nickel screens gave rise to fiber production (Fig. l a ) bers in the literature. In addition, this study deals if the molar ratio of coppcr to cystine was in the neighborwith the stoichiometric and other properties of the hood of unit.y. I f the ratio dcviated from unity by a factor of ten in cither d i r e d o n , the fibers became very scarce. copper complex in solution. These tests indicatcd that t.he cystine diffused through the Copper-cystine (CuCsHlON20&32) in the form of formvar film and reacted with coppcr ions to form a copperneedles \vas prepared and analyzed for copper con- cystinc compound. tent about 50 years a g ~ Ray . ~ and ~ Sen5 ~ ~msl,de ~ Measurements of Size.-The lengths of the fibers seem to determinations of magnetic moment on 33 copper be a function of the method of preparation and treatment, solutions giving fibers of shorter lengths. The complexes including a copper-cystine, ( C U C ~ H ~very ~ - dilute dianietcr of the fibcrs was determined by methods previously N.,0&32.2HzO). Numerous studies of complex for- described for tobacco mosaic measurement .I4 For measuremation between copper and other amino acids, no- ment of fiber widt,h the most, accurate results were obt,ained tably g l y ~ i n e , ~ -have ' ~ emphasized the change in by nicasuring across a group of contiguous fibers and dividing by the number of fibers, while for t,hc determination of type of complex formation with change in pH. height,, shadow methods were used. However, while Materials.-An aqueous solution of cystine (Nuhitional Biocheinicals Corporat.ion) was made up 0.000425 molar, which is slightly lcss than saturated, as a stock solution. The copper scrcens (Li!ktro-mesh) as used in t,he following elect.ron microscopic osperiments appeared to be coated with a light superficial film of c,opprr salts but no att,empt, a t analysis was madc. The nickel screens (C..O. Jelliff 1Ifg. Corg.) were spect rographically analyzed (Rlincral Tachnology Division, P. 31. Ambrose, Chief, U. s. Bureau hIines). This disclosed thc presence of 1% copper and less than 0.0570 chroinium. Experimental.-Drops of the cystine solution were put on copper screens coatcd with forlnvar and after being dried were found by use of the elect,ron microscope (type E I I U ) to give rise to the charact.erist.ic hexagonal plates of cystine and R largt! number of fine fibers of high density, usually occurring in clumps. (Similar results y ~ r eobt.ained when the cyst.ine was recrystallized four t,imes from water, and from a specially purified preparation kindly donated by Dr. J . P. Gi~enstcin.) When drops of the same cystine solut.ion werc dired on nickel screens no fibers were observed. Fibers were also obtained by immersing. a copper wire in a ( I ) Public Health Service, National Institutes of Health, Federal Serurity Agency. ( 2 ) G . Embden, 2. p h ~ ~ s i oChem., l. 32, 94 (1901). (3) J. hIaiithner. Z . Biol., 42, 170 (1901). (4) C. Neuberg and P. RIayer, Z . p/i2/siol. Clrear., 44, 502 (1905). ( 6 ) P. Ray and D. N. Sen, J . I n d i a n Chem. S o c . , 25, 473 (1948). (0) A. Albert, Biorhem. J . , 4'7, 531 (1950). (7) J. Rjerrum. "Rletal Anitnine Formation in Aqueous Solution," Copenhagen, 1041. (8) 11. Uorsook otid I
