The Effect of Urea on the Electrophoretic Patterns of Serum Proteins

Preparation of Monoalkylamino Alcohols.-Molar quan- tities of the alkyl bromide and 2-amino-2-methyl-propanol-. 1 were refluxed in ethanol solution fr...
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TABLE I -Monoalkylamino

K -

M. p., 'C.

alcohols (CHsC(NHK) (CHa)CHpOH)-

B . p., 'C.

Ethyl 75.5-76.5 162-163 %-Propyl 59.5-60.5 183-185 &Propyl 43.0-45.0 165-166 n-Butyl 69.5-70.0 195-196 i-Butyl 51.0-52.5 185-186 n-Amyl 60.0-60.5 210-217 i-Amyl 176.5-77.0 205-207 a Discordant results probably due to

Mol. formula

Nitrogen, % ' Calcd. Found

CeHisON 11.95 C,HllON 10.67 C7HlrON 10.67 CsHlsON 9.64 CaHgsON 9.64 CsHsoON 8.80 C ~ H ~ O O N 8.80 persistent impurities.

11.91 10.55

10,64 9,48

9,70 8.78 8.76

-9-Nitrobenzoates M. p.,

OC.

206.5-207.0 185.0-185.5 140.0-14 1 . 0 163.b 1 6 4 . 0 165.0-166.0 151.0-151.5 168.0-168.5

of monoalkylamino alcoholsMol. Nitrogen, % formula Calcd. Found

CiaHiiOaNz 10.52 C1cHzoO4Nz 9.99 C~dHtoO4Na 9.99 CisHuOrNn 9.52 CisH220cNa 9.52 C i e H ~ ~ O ~ N 2 9.09 C1aHzlOlX2 9.09

9.75

' 9.22 9.27 9.03

9.10

non-reproducible results. The nitro esters were very pale yellow solids. The p-aminobenzoates can be Obtained by reducing a paste of the nitro ester in concentrated hydrochloric acid with powdered tin. The temThe. p-aminoperature must not exceed 40-45'. benzoic acid ester hy&ochlorides are then prepared by treating an ether Of the free base with gaseous hydrogen The hydrochlorides are white, extremely hygroscopic solids. The physio~ogica~ effectof various derivatives Of the anesthetic bases be reported

layer was drawn off and the oily layer transferred to a beaker, where it rapidly solidified. The rnonoalkylamino alcohols can be purified by recrystallization from petroleum ether. Melting points and analyses of the alcohols are given in Table 1. Preparation of the p-Nitrobenzoic Acid Esters.-To 14.5 g. (0.1 mole) of the monobutylamino alcohol dissolved in 50 ml. Of freshly distilled pyridine, was added 18.6 g. (0.1 mole) of p-nitrobenzoyl chloride. The addition was made in small portions and with constant stirring. Care is necessary in maintaining the temperature between 30 and 40'. After addition of the p-nitrobenzoyl chloride was completed, the mixture was allowed to stand for twenty-four hours and then diluted with 400 ml. of water. A voluminous curdy yellow precipitate formed and was filtcrcd off, washed with 80 of 2% sodium carbonate

Experimental

solution and allowed t o dry. The nitro ester thus obtained was purified by recrystallization from ethanol. Melting points and analyses of the nitro esters are given in Table I.

The 2-amino-2-methyl-propanol-1 was purchased from the Commercial Solvents Corporation, and the alkyl bromides from the Eastman Kodak Company. All of the materials were purified by redistilling. Preparation of Monoalkylamino Alcohols.-Molar quantities of the alkyl bromide and 2-amino-2-methyl-propanol1 were refluxed in ethanol solution from fifteen to fortyeight hours. The ethanol was then distilled off and the residue treated with 30% sodium hydroxide solution. The warm solution separated into two layers. The aqueous

[CONTRIBUTION FROM

THE

Summary 1. A series of new monoalkylamino alcohols is reported. 2 . The p-nitrobenzoates of these amino alcohols have been prepared. NEWYORK, N. Y. RECEIVED JANUARY 30, 1942

ELECTROPHORESIS LABORATORY, COLLEGE OR PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY]

The Effect of Urea on the Electrophoretic Patterns of Serum Proteins BY DANH. MOORE The effect of urea on the electrophoretic pat- aqueous solutions. The patterns depending upon terns of serum proteins has been examined in the refraction gradients at the protein boundaries electrophoresis apparatus of Tise1ius.l One of were obtained by means of the scanning method the important characteristics of the Tiselius of Longsworth.2 method is its ability to reduce convection in the Normal human serum was diluted 1:4 with a cell (U-tube) caused by heat generated by passage 0.02 M phosphate buffer containing physiological of current. This is done by submerging the ap- saline and 2.5 M urea, and dialyzed against the paratus in a bath which should be maintained at same buffer containing the urea and saline. The the temperature where the change of density with electrophoretic pattern is illustrated in Fig. la. temperature is least, i. e., about 4" for dilute The albumin is apparently broken up into three (1) A. Tiaclius, Trams. Faraday SOC.,88, 524 (1837).

!21 I.. G . Longsworth, T R I S JOVaNAL, 61,

629 (1989).

May, 1942

I

.'1

. I.

(b)

6

(C)

.~ Carded stem*the Of the Stem being at both ends. The stem was ground into the neck of the bulb to give a good ground glass fit. The bulb and neck were filled 13)

L

(i

Lonolwo&

D A M . c I ~ ~ - . CI.-

R-, ad. 271

with the solution whose change in density with temperature was to be measured and cooled to a low temperature before the thermometer capillary was inserted. A drop of dye was added to the open end of the capillary and theshulh was further cooled to draw the dye down in the stem and form an

I1 ~

-

temperature of 1.5", which has been shown' to he about the correct temperature for dilute aqueous buffer solutions. In order to determine whether the pattern illustrated in Fig. la is the result of convection, the change of density with temperature in the region of 0" for the urea solution was studied. An improvised dilatometer of high sensi-

(1939,

1091

UREAON ELECTROPHORETIC PATTERNS OF SERUM PROTEINS

-

-

3

-

-

2-

I

-

1 1 1 1 1 1 1 1 1 1 1 1 1

-

-

Fig. Z.-Density.-tempersturee curves for some common aqueousrolutions: (1) water. (2) 0.05 Mlithium barbiturate 0.025 M LEI. (31 0.15 M NaCl 0.02 .M sodium~~, ohmphate. (4) 1.0M %a, (5) 1.8 Ai SUC& (6)2.8 M urea 0.15 M NaCl 0.02 M sodium phosphate. (7) water.

+

+

.

+

~~~

+

Densities ere relative, consequently the rloper of the end not their positions ere sipoiiimnt.

NW*I

I002

DANH. MmnF:

Fig. 3.- l ~ l ~ r ~ r o palirrin ~ ~ l ~ of ~ ~iiorinal r ~ ~ humaii ~ i ~ serum in 0.02 .lf s ~ t l i i i i i iphosphale bufier containing 0.15 .V NaCl at p1.I 7.4 i i i a temperature bath st 17'. L e f t ascending pattern. right descending pattern.

VOl. 64

3 which was obtained in phosphate buffer without urea but in a bath a t 17O instead of the usual 1 3 . This pattern is similar to that of Fig. l a but is not identical. Even a t this high temperature the slope of the density-temperature curve (curve 7, Fig. 2) is not as great as that of the 2.8 M urea solution (curve G). These experiments illustrate the importance of carrying out electrophoresis analyses at a temperature where the change in density with temperature is small: For dilute aqueous solutions which are represented by curves 2 and 3 of Fig. 2, this temperature is between 0 and . '4 Protein concentrations of 2 or 3% do not shift the temperature of maximum density appreciably so that a bath temperature of 0 to 2' is ideal for general electrophoretic analysis. I wish to express my appreciation to Mrs. Vernon Strub and the Misses Helen Sikorski and Evelyn Kulp for their contributions of sera and technical assistance, and my indebtedness to Dr. H. A. Abramson of Columbia Univcrsity and to Dr. L. G. Longsworth of the Rockefeller Institute for their valuable suggestions and interest.

pcrature. Solutions of ordinary dilute buffers give almost a horizontal line between 1 and 6'. The 0.02 d l phosphate buffer with 0.15 M sodium chloride (curve 3) has a maximum density of about 1.3' but the curve is still comparatively ' 4 The curves for the more con=horizontal at . trated urea and sucrose solutions have steep slopes a t temperatures as low as -4', which SummarY would result in marked convection if the heat dis1. Electrophoretic patterns giving a large sipation in the electrophoresis cell were not kept a t a low value. I t is observed that curve 6 number of components for human serum in the urea !I have been shown to be a begins to flatten out a t about -5'. An electrn- presence of 2.8 -\ phoresis experiment was carried out in a bath at result of convection currents in the electrophoresis -7O whereupon the serum with 2.8 M urea gave cell. The change of density with temperature for only slight indications of the anomalies found in a urea solution of this concentration is compara' . Fig. la. If the voltage were dropped to 2.4 tively large in the region of 0 to 4 2. Density-temperature curves for a few comvolts/cm. (half the original vaiue), no effect of urea was observed. These experiments lend mon solutions are given. N. Y. RECENEDF E E R U 14, ~ Y1942 evidence to the belief that the jagged pattern is NEWYORK, a resilt of convection. Another indication that (4) Data om lem-turr 01 maximum d t a 6 t y I a number 01 the pattern of Fig. l a was caused by convection .oIu:iom m a y be l o u d i n the "lateml.tiond Critical T.bl-:' Vol. 111. MrCnr-Hill B m k Company. loc.. F e r Vork. N. Y..1929. is the pattern for normal serum illustrated in Fig. P. 10s.