Hapten Inhibition of Precipitation of Antisera Specific to the p-Azophenylarsonic Acid Group, the p-(p-Azophenylazo) Phenylarsonic Acid Group, and the p-Azophenylmeylarsinic Acid Group Citation Pardee, Arthur Beck (1943) Hapten Inhibition of Precipitation of Antisera Specific to the p-Azophenylarsonic Acid Group, the p-(p-Azophenylazo) Phenylarsonic Acid Group, and the p-Azophenylmeylarsinic Acid Group. Master's thesis, California Institute of Technology. doi:10.7907/ga0t-gq17. https://resolver.caltech.edu/CaltechTHESIS:03192025-182405303 Abstract No abstract. Item Type: Thesis (Master's thesis) Subject Keywords: (Chemistry) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Pauling, Linus Thesis Committee: Unknown, Unknown Defense Date: 1 January 1943 Record Number: CaltechTHESIS:03192025-182405303 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:03192025-182405303 DOI: 10.7907/ga0t-gq17 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17073 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 20 Mar 2025 22:18 Last Modified: 20 Mar 2025 22:18 Thesis Files PDF - Final Version See Usage Policy. 3MB Repository Staff Only: item control page

Hapten Inhibition of Precipitation of Antisera Specific to the 2-Azophenylarsonic Acid Group, the :e-(.E.-Azophenylazo)phenyla.rsonic Acid Group, and the .E-Azopheeylmethylarsinic Acid Group. Thesis by Arthur B. Pardee In Partial Fulfilment of the Requirements for the Degree of Master of Science, California Institute of Technology, Pasadena, California. Hapten Inhibition of Precipitation of .Antisera Specific to the 11-Azophenylarsonic Acid Group, the .E'"" ~ .A zophenylazo)phenylarsonic Acid Grou:-_p, and the .l?"'Azophenylmethylarsinic N:id Group. This work was carried out as a part of the Innnunochemistry program at the California Institute of Technology. A series of compounds similar to phenylarsonic acid were prepared for the purpose of examining the effect of changes in the structure of the acidic group on the haptenic action. Previous work has been done by Erlenmeyer and :13erger1 who found that either ovalbumin or horse serum globulin coupled with diazotized :p-aminophenylphosphonic acid gave precipitates with antiserum homologous to the :e,-azophenylarsonic acid group. However proteins coupled with diazotiz.ed :e,- aminophenylstibonic acid did not react with this serum. Eerinl Hawowitz and 2 tested the inhibition of sixteen simple, easily available sub- stances. inclU:ding phenylphosphonic acid, on the precipitation of an anti• azophenylarsonic acid serum. sually. The amount of inhibition was estimated vi- Phenylphosphonic acid showed good inhibition, but the other sub- stances. most of which were not very similar in structure, did not. Gross- berg (unpublis~ed results) found that benzoic and benzenesulfonic acids inhibit poorly. ~iE1~e1;t~ Methe£! Pre;e8;!'~~2n~ of CoLI!e.9~~ Ph~,l,1=.ax:.~q_nic ~acid.Arsanilic acid.- --._,, • ....r,A r, _ ...,...._ Prepared by D. Erown .. Prepared by G. Cleland · acid.- Prepared by the method of Michaelis .Phenylphosphonic . -1/J H-~~---------+ PC1 A1Cl PO1 Cl > r/J PC1 H0 PO H 3;> 3 (; 2 2 4 2 ?' 3 2 A mixture of 60 ml. of phosphorus trichloride, 180 ml. of benzene and 16 g. of anhydrous aluminum chloride was refluxed in an all glass apparatus on an oil bath for 4o hours. The mixture turned dark and two phases appeared. The layers were separated, and the top layer was distilled up to 850 to eliminate unchanged. phosphorus trichloride and benz ene. ml.) was extracted with 50 ml. pet. ether. 0° , and was saturated with chlorine. The residue (30 The ether layer was cooled to A white precipitate appeared. was careful~ added, drop by drop, at 0°. Water A very vigorous reaction ensued. Aqueous sodium hydroxide was added till the solution was just blue to congo red, the solution was evaporated to 75 cc. to be two compounds present. and cooled. The less soluble was phenylphosphonic acid. The product was recrystallized twice from water. M.P. 158° C Yield 0.5 g. 4 Prepared as described by Christiansen • ~!,&_lst~i-.a•amounts were used. There appeared Half The final precipitate waz extracted with alcohol, then with acetone, washed with water and dried. Yield 36% "Met_&larp.£Q_ic .fl.~•- Prepared by w. Renfrow. Prepared by the method of Quick and Ad.ams 5• f CH2Cl + Asa03 '/> CH2AsO3H > .A mixture of 12.6 g. benzyl chloride, 5 g. arsenious oxide, and 15 cc. of lON aqueous sodium hydroxide was refluxed with stirring for two hours. upper layer was separated and discarded. The lower layer was made just neutral , and the slight precipitate which came down was discarded. was added till the solution became just blue to congo red. Yield 1.7 g. Acid The precipi- tate was immediately filtered off, washed with cold water and dried. M.P. 166-168°. The ~ull2.e!fil}N s9n i c ~.1£-- Prepared by the method of Bertheim6. f As03H2 I~o~cf <f>AsCl2 H:aO )' f Aso c~~I -;,jAJH3As02Na _ H_+~~f1JH3As02H A solution of 10 g. of phenylarsonic acid and 0.1 g. potassium iodide in 50 ml. of 12 ! hydrochloric acid was saturated with sulfur dioxide at oo for l hour. The dichlorophenyl arsine separated as a heavy oil and was added to 100 ml. of water containing 15 g. of sodium carbonate. The phenylarsine oxide thus formed was dissolved in 8 ml. of 10 N sodium ey. droxide solution to which was added 32 ml. ethanol and 4 ml. methyl iodide. The progress of the reaction was followed by withdrawing a sample and titrating with I 3 - in neutral solution. hours. The reaction is complete in two 100 ml. water was added and this was extracted with 20 ml. ether. The ether was discarded. With stirring , 8 g. of silver nitrate in 20 ml. of water was added to remove the iodide ion. As soon as a drop of acid gave no iodine color, 20 ml. of concentrated nitric acid was added. ver iodide was filtered off. Sil- Then 10 g. more of silver nitrate in 20 ml. water was added and the solution was just neutralized with ammonium hydroxide. The silver phenylmethylarsinate was filtered off. added to 8 ml. 6 N hydrochloric acid in 150 ml. water. The salt was When all the pre- cipitate seemed to have become silver chloride, the filtrate was evaporated to dryness on the water bath after neutralization to congo red with ammonium hydroxide. The residue was extracted with 10 ml. hot alcohol, , filtered , and evaporated to d:cyness. This residue was dissolved in 1 ml. water and reprecipitated with warm acetone. twice. Yield O. 5 g. The precipitation was repeated 6 the method of Eertheim. F\. (a) -H+ NH2'\._/AsOi ~ 10.8 g. (.05 mole) arsanilic acid was dissolved in 80 ml. 12 N hydrochloric The solution was saturated with sulfur dioxide at 250 c. acid. of potassium iodide in 3 ml. water was added with stirring. After a few minutes the entire contents became almost solid and was filtered. residue was added to 6 g. sodium hydroxide in 50 ml. water • droxide was added in excess to the hot solution. 0.1 g. The .Ammonium ey- On cooling, .E,-amino- Yield 90%. phenylarsine oxide crystaliized out. 0.06 mole of the J?_-aminophenylarsine oxide was dissolved in 12 ml. 10 N sodium hydroxide. 4 ml. of methyl iodide was added. The mixture was shaken until the titer with triiodide ion in neutral solution became small. This required about two hours. Freshly prepared silver chloride from 8 g. silver nitrate was added with stirring until a drop of acid gave no iodine color. Then 20 ml. of 6 N eydrochloric acid was poured in, mak- ing the solution blue to congo red. were filtered off. The silver chloride and silver iodide On evaporation to about 4o ml. an orange color appeared. The solution was dried in a desiccator over calcium chloride and the resulting solid mass was extracted. with 50 ml. alcohol and treated with ani1!!81 charcoal. After filtering off the charcoal, 100 ml. isopropyl ether was adde<i to the alcoholic solution. A light colored solid separated. It was redissolved in aleohol and reprecipitated. Crystals were obtained by dissolveing in a minimum of alcohol. adding a large excess of nitrobenzene, and allowing the solu.tion to evapo~ate 1low:cy. Yield 31%•. M.P. 201° droxide .... Methyl Grignard was made from 30 g •• methyl iodide by the usual method. 0.1 mole dichlorophenylarsine in 30 ml. ether was dropped in with stirring. at 0° c. After refluxing 1/2 hour, ic·e and dilute HCl were added. The ether phase was separated, and the ether distilled off. dimetbylarsine was distilled at 60 mm. E.p. The phenyl- 13507. To this point, starting with phenylarsonic acid, the yield was 4o%. The phenyldimethylarsine was dissolved in 150 ml. petroleum ether (80-100°) and 10 ml. water was added. A 25% bromine solution in petro- leum ether was added drop-wise with shaking, until there was a very faint yellow color. The water leyer was s~parated and evaporated to 3 ml. brownish solid which came out was dissolved in 30 ml. hot acetone. insoluble residue was filtered off and discarded. tion, thick, pale broTl'Il crystals came out. The The On cooling the solu.- On recrystallization from 5 ml. water and 30 ml. acetone, the hydroxybromide melted at 161-162°. Yield, 0.6 g. An aqueous solution of the eydroJcybromide was shaken with excess sil• ver ox1'd e8• The solid was filtered off, and the solution of the dehydro- xide was used directly. ~i,?,~__ion~t_?n,l gf fhenllme~&l~inj.p ~i~.§l.t.£1•,2.0 ~ A portion of 0.0103 g. of the acid was titrated at 25.5° c•. in 20 ml. H2 0, with 0.025 N Na.OH. A Beckman ~del G. pH meter was used. ml. were required for complete neutralization. 2.00 - 6- Data: ml H+ x 10 6 Ratio :: = ml. NaOH 2- ml. NaOH 10 6 x K = (H+) NaOH pH o.6 5.32 4.8 o.43 2.11 0.7 5.40 4.0 0.54 2.16 0.85 5.54 2.9 0.74 2.15 1. 00 5. 68 2.1 1.00 2.10 1.15 5.82 1.52 1.35 2.05 1.30 5.96 1.10 1.86 2.05 f~1 6 x 10 2.1 From the inflection point pK = 5.65. -6 This gives K = 2.2 x 10. As 0 3 H2 was similarly found to be= 3.7. pK of This result was less accu- rate than the previous one because too dilute a solution was used. Press- man and Brown9 found pK = 3.5 for this acid. Ex erimental Procedure for Inhibition ~Tes.t s. Two separate sets of hapte.n inhibition tests were made with two different sera. One cc. each of hapten, and of R 1 ~ ~ ! (antigen H03S ~ / S 0 3 H (R' = -?rN Q l[N Q XXX) As0 3 H2 ) in a buffer of 0.16 M boric acid con- t:aining 0.9% sodium chloride at pH 8.0 were mixed and one cc. serum was added. This mixture was allowed to stand at room temperature for one hour, and then for approximately 24 hours in the refrigerator. The cen- trifuged preci~itate was then analyzed by the method described by Pressman 10 • - 1- Analyses were run in triplicate. Average deviation from mean was less than 2.5% . pH of the supernate, unless otherwise stated, lay between 7.9 and 8.1. Thanks are due to Mr. Grossberg for help with these analyses. ~t,a: • Set_No:1• Serum: -R serum) undiluted. .Anti-azophenlarsonic acid sheep serum. (anti Antigen diluted 1/26,000 for maximum precipitate. Values are tabulated as fractions poF mille of the amount precipitated. in absence of hapten, 230_,µg• p~ta_; §~_No: ~• Serum: .Anti-azophenyl-azophe:eylarsonic acid sheep serum, (anti-R 1 serum) diluted to 1/2 concentration. 1/40,000 to obtain maximum precipitate. Antigen diluted Values are of the amount pr&- cipi tated in the absence of hapten, 169"-g. pa~,€!,: Se~ Ng. ~- Inhibition by some inorganic salts. Conditions were the same as in set No. 1 pH 8.0 - 8.1. In tripli- cate. Set No.~,.,_, 4. ., ,.., __ Four ·rabbits were injected with p-,azophe:eylmethyl,.. arsinic acid sheep serum. Three produced anitbodies. were pooled and used for inhibition tests. 2 cc. These three sera of serum and testing antigen p.-azophen,ylmethylarsinic acid ovalbumin were mixed and varying amounts of hapten in 2 cc. of buffer at pH 8.0 added. room temperature then 24 hours at 0° c. Stood 1 hour at Values are tabulated as frac- tions poP mill~ of the amount precipitated in the absence of hapten, Data: Set No. 1 Inhibition by Haptens of the Precipitation of .Anti -R Serum .Antigen solution, 1 ml. (38/(-g ); hapten solution, 1 ml.; antiserum, 1 ml. pH of supernates, 3.1 Moles hapten added XlO 3 1.4 4.1 123 37 111 333 1000 Fract. amount of precipitatea :E,-Aminophenyla.rsonic acid o. 97 0.31 0.58 0.34 Phenyla.rsonic acid 1.00 0.90 0.67 Phenylphosphonic acid 0.99 0.90 0.39 o. 77 0.50 0.17 Eenzyla.rsonic acid 0.95 0.89 0.77 Phenylstibonic acid 0.99 0.95 :2,-.AminophenylmethylBxsinic acid 0.65 o.47 1.02 0.98 0.95 0.80 0.61 Phenylmethyla.rsinic acid 1.00 0.92 o.66 Metbyla.rsonic acid 1.00 1.03 1.00 0.91 0.72 Phenyldimetbylars ine hyd.roxybromide 0.97 1.01 0.96 :2,-.An:.inophenylarsinoxide a Amount in absence of hapten, 23o/-(.g• b pH, 8.2 0.93b 1.02 0.98 0.98 0.99 0.97 Data: Set No. ,2 Inhibition by Haptens of the Precipitation of Anti -R 1 Serum .Antigen solution, 1 ml. (25_,.Lg); hapten solution, .1 ml.; antiserum, diluted with 1 vol. buffer, 1 ml. pH of supernates, 8. 1 Moles hapten added Xl0 1.4 4.1 123 37 111 8 333 1000 Fract. amomt of precipitatea o.43 ]:--.Aminophenylarsonic acid 0.74 0.58 Phenylarsonic acid 0.90 o. 72 0.51 Phenylphosphonic acid 0.90 0 .86 o. 61 o.42 Eenzylarsonic acid 1.09 Q.88 o.88 0.27 Phenylstibonic acid 1.12 1.05 o. 16 1.11 0.99 ,E.-.h.minophenylmethylarsinic acid• 1.03 0.95 Phenylmethylarsinic acid 1.00 0.96 0.97 0.93 0.78 Methylarsonic acid 1.03 Phenyldimetbylarsine bydroxybromide ,E.-.Aminophenylarsinoxide a Amount in absence of hapten 16~. 1.07 0.90 0.78 1.08 0.95 0.93 0.77 1.03 1.03 1.06 1.05 Data: Set No. 3 Inhibition by Inorganic Salts of the Precipitation of .Anti -R Serum .Antigen solution, 1 ml. (3~); salt solution, 1 ml.; antiserum 1 ml. pH of supernates 8.0 - 8.1 Moles added XJ.05 Salt l 10 Fract. amount of precipitatea N~HA.s04 0.92 o.44 Na.As02 1.16 0.40b NaaHPQ 4 0.94 .40 Na2S0 4 . .95 .79 1.00 NaCl a .Amount in absence of added salt 230~. b pH of supernatant, 8. 4. Data: Set No. 4 Inhibition by Haptens of the Precipitation of Antiw azophenylmethylarsinic .Acid Serum .Antigen solution, 2 ml.; hapten solution, 2 ml.; antiserum 2 ml. pH of supernates, 8.1. Moles of hapten added no8 1 3 10 100 1000 Fract. amount of precipitatea Phenylmethylarsinic acid 0.93 0.81 0.73 o.oo o.oo Methylarsonic acid • 79 .64 ~(Jl-Hydroxyphenylazo)phenylarsonic acid .98 .71 Phenylarsonic acid • 93 • 90 • 57 Benzylarsonic .acid .95 1.03 .68 Phenylphosphonic acid 1. 03 • 69 a Amount in absence of hapten, s7g. 1. 05 • 53 ----------Conclusions 1. Substitution of -CH3 for -OH on the end group has a far greater effect than has substitution of groups onto the ring. have a still greater effect. Two substitutions It is a question as to whether this is a steric or a charge effect. CH3 A~0 2 H to about the same ex,. 0 As0 3 H2 inhibits anti NN Q CH3 tent as <:::)As0 2 R inhibits anti -lrn Q 2. As0 3 H2 • This indicates that the effect is not entirely steric. 3. ,CH3 Methyl arsenic acid, which has both the groups - As0 2 H and -Asg:H' inhibits anti - lrn Q OH As~!~ fairly well, but anti NN { )As0 2 H rather poorly. . 92H, ys!½ 4. Steric effects could be tested by using Q 5. Substitution of P for As leads to no appreciable change in the haptenic strength. However, a change of Sb for As causes the compound to have no inhibiting effect. noted fact that A::s0 2 H and Q As0 2 H. CJ Sb03 H2 This can be correlated with the previously i s really (3 0 Sb02 •H 2 0) 2H2 0 .with two easily removable waters. 1. Some compounds similar to z:) As0 3 H2 hibiting action. 2. have been studied for in- References ---.......,,..____. (1) Erlenmeyer and :Berger, :Biochem. z., ~ ' 429 (1932). (2) Haurowitz and Ereinl, (3) Michaelis, :Ber., 6, 819 (1873). ~ Christiansen, 11 0rganic Derivatives of .Antimony 11 , New York, (4) z·. Physiol. Chem., m, 111 (1933). Chemical Catalog Co., 1925. --- (5) ~uick and Adams, J.A.C.S., 44, 811 (1922). (6) :Bertheim, :Ber., 48, 352 (1915). (7) Winmill, J. Chem. 101, 723 (1912). __.,.. . Soc., -..,,,,...,,.., (8) Steinkopf and Schewn, ~ - , ~ ' (9) Pressman and Brown, J.A.c.s., (10) Pressman, Ind._Eng. ,Che,!E•,,1., Anal. :Ed., ~• 357 - ~- 2795 (1921). £5., 54o (1943). (1943).