Improved Bacterimetric Method for Cystine GERALD D. SHOCKMAN, JOSEPH J. KOLB, and GERRIT TOENNIES Lankenau Hospital Research Institute and Institute for Cancer Research, Philadelphia 7 7, Pa.
A modification of the bacterimetric method for determining cystine increases sensitivity, reduces incubation time, and gives a precision of 2 to 3% on hydrolyzed ni aterials.
B
ACTERIMETRIC cg,stine determinations suffer from the difficulties of high blanks (due to color and/or cystine contaniination of the media). long incubation times, irregular assay vurves, lack of specificity, and lack of sensitivity when compared with bacterimetric assayt for other amino acids. This lsck of sensitivity is not consistent with the low cystine content (1,4 ) of the organisms used to asbay the amino acid and is a great disadvantage in assaying hydrolyzates low in cystine. .is a result of experimentation with several published procedures, the method of Riesen et at. ( 3 ) ,using Leuconostoc mesentermdes I’D-60 in their oxidized peptone medium, has been modified as follow.:
A . Their medium P:a.AH was used, without xanthine, with tMire the Dbmethionine and rpproline and four times the DLtryptophan concentrations, and with Gtyrosine and basparagine added in concentrations of 400 and 200 mg. per liter of finished medium. This was prepared as double-strength basal medium, with glucose added aseptically after both solutions are autoclaved separately for 3 minutes a t 15 pounds’ pressure. Separate glucose sterilization gives low and stable color blanks. R. Ascorbic acid (250 mg. per liter) was included in the medium. This highly reducing environment greatly increases the speed and amount of growth response to cystine. C. Inoculum was grown several times in the assay medium i n the presence of 0.4 y of cystine per ml. (twice as much as the highest standard level), arrested in the logarithmic growth phase and used without washing, at the rate of one drop per tube (18 X 150 mm.) containing 6 ml. of finished medium (6). D. Absorbance measurements rather than titration of acidity were used. Procedure* C and D help to reduce incubation time and improve the precision of the assay. E. -411 samples and standards ale made up to the same volume
( 3 ml.) and autoclaved for 3 minutes a t 15 pounds. No cystine destruction under these conditions has been noted. The same volume of double-strength basal medium (including the separately autoclaved glucose) is added aseptically, and tubes are inoculated and incubated a t 37’ C. overnight. Two sets of readings, a t approximately 17 and 22 hours, have been found helpful in detecting nonspecific responses or effects of the substance being assayed.
Typical Readings on Set of 8 Tubes Cystine, y/ml. finished medium Readingsa At 17 hours
At22hours 0
103 95
_.IIY
0 150
0.075
0
81
319 320 340 338
49 1 $98 531 339
0.225 658 652 678 678
Adjusted optical density (6).
In the authors’ hands, these modifications increased the sensitivity of the assay approximately fiftyfold, from a maximum level of standard of 11 to 0.22 y per ml., reduced the incubation time from 72 to 17 hours, and gave a precision of 2 to 3% on hydrolyzed materials. Under certain hydrolysis conditions cystine niny be partially destroyed ( 9 ) . LITERATURE CITED
(1) Camien, AI, K.,Salle, -4.J., and Dunn, .\I. S., Arch. Biochem., 8, 67 (1946). (2) Kolb, J. J., and Toennies, G., ANAL.CHEM.,24, 1164 (1952). (3) Riesen, W. H., Spengler, H. H., Robblee, A. R., Hankes, L. V., and Elvehjem, C. A., J. B i d . Chem., 171, 731 (1947). (4) Shockman, G. D., and Toennies, G., Arch. Biochem. Biophys., 50,9 (1954). ( 5 ) Toennies, G., and Gallant, D. L., J . B i d . C h a . , 174, 451 (1948). RECEIVED for review -4pril 10, 1964
Accepted .June 19, 1954.
Rapid Identification of Glycols in Alkyd Resins CHARLES 8. JORDAN Paint and Chemical Laboratory, Aberdeen Proving Ground,
,\ rapid method, which utilizes only standard labora-
tory equipment, has been developed for qualitatively detecting vicinal glycols of low molecular weight in alkyd resins. It is based on reflux distillation, making use of a ternary azeotropic mixture formed by xylene, the glycols, and water, xylene being the refluxing medium.
T
HE polyhydric alcohol components of alkyd resins have a bearing on the perforinanre of the end product. No suitable procedure for the identification of vicinal glycols in alkyd resins containing three or more polyhydric alcohols was available. Orchin ( 3 ) developed R procedure which is applicable to the analysis of polyhydric alcohol fractions containing ethylene glycol, propylene glycol, diethj lene glycol, and/or glycerol, utilizing the well-known periodate scission of adjacent hydroxyl groups. By making use of standard procedures he was able to identify the resulting oxidation product$. The fact that some alkyd
Md.
resin components, such as sorbitol, mannitol, or pentaerythritol, interfered in some cases with Orchin’s procedure suggested the need for a method of separating the polyhydric alcohols. During the development of the Jordan and Hatch ( 2 ) procedure for the determination of water in glycols and glycerol, several solvents were found which formed ternary azeotropic mixtures with ethylene glycol and water, including benzene, toluene, xylene ( 6 ) , petroleum naphtha, cyclohexane ( 4 ) , and turpentine. For separating glycols of low molecular weight from other polyhydric alcohols, xylene was found very satisfactory as the refluxing medium in the Dean and Stark ( 1 ) reflux distillation procedure. The presence of the vicinal glycols after this separation could then be verified by the standard qualitative periodate procedure. Shay et al. (,5) recently published a useful procedure whereby individual polyhydric alcohol components of alkyd resins, or binary mixtures of these alcohols, could be identified by infrared spectrophotometry. Because three or more polyhydric alcohols
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