The Effect of Urea on the Sedimentation Coefficient of the Curly Top Virus Dimer

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Utah State University DigitalCommons@USU Undergraduate Honors Theses Honors Program 1976 The Effect of Urea on the Sedimentation Coefficient of the Curly Top Virus Dimer Allen H. Smith Utah State University Follow this and additional works at: http://digitalcommons.usu.edu/honors Part of the Plant Sciences Commons, and the Virology Commons Recommended Citation Smith, Allen H., "The Effect of Urea on the Sedimentation Coefficient of the Curly Top Virus Dimer" (1976). Undergraduate Honors Theses. 200. http://digitalcommons.usu.edu/honors/200 This Thesis is brought to you for free and open access by the Honors Program at DigitalCommons@USU. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of DigitalCommons@USU. For more information, please contact dylan.burns@usu.edu.

'Ibe effect of urea on the Sedimentation Coefficient of the Curly Top Virus Dimer by Allen H. Smith

/ The effect of urea on the Sedimentation Coefficient of the Curly Top Virus Dimer. I. Introduction The purpose of this project is to investigate the effect of increasing concentrations of urea on the sedimentation rate of the curly top virus dimer as measured by ultracentrifugation techniques. In general, urea causes the breaking of h~drogen bonds in macranolecules--in the case of the virus, increased concentrations of urea should cause changes in the configuration of the virus and may possibly cause a separation of the dimer into monomers. From previous centrifugation studies 1, we know that the dimer has a characteristic sedimentation coefficient(s) of about 80 Svedbergs; the monomer has an S value of about 55 Svedberg units. Increased concentrations of urea should cause increased changes in the configuration of the virus particle with a concomitant decrease in the sedimentation coefficient. II. Experimental Band sedimentation technique 2, utilizing a type II band-fonning centerpiece3 was used to determine the S values. A 20 ml sample of curly top virus solution was sedimented through gradient solutions containing a constant concentration of cesium chloride (.35M), but containing varying concentrations of urea. Eight runs were made under the following conditions:

2 Run Tanp. oc RPM Sector (. 5&nl ) sv 224 20 24,000. 35M CsCl + a.1 urea. Sv 228-1 11 11 II.25-2 II II II.50 sv 229-1 II II II.25-2 II II II.50 sv 230-1 " -2 " sv 231-1 " II II.90 11 11.70 II II 1.0-2 II 11 11 1.8 sv 232-1 11 II 11.10-2 " 11 II.20 sv 234-1 11 II 11 2.8-2 " " sv 235-1 " -2 " 11 11 3.7 ".20 II 11 0 s 73 73 72 59 59 59 61 57 51 70 72 43 35 73 74 I I I. Calculations: In order to compare seciinbntation coefficients, differences in viscosity and density due to added urea must be minimized by applying corrections that theoretically yield an S-value for a gradient solution having the density and viscosity of water at 20 C (S ). Following is 20 'w. the equation used: s 20,w = sn r c1-vc\v) c1-vd) where S = sedimentation coefficient obtained fran the experiment d = density of water w d = density of gradient solution v = specific volume of the virus (cm 3 g-l) n = viscosity of the solution relative to water r S = theoretical coefficient in water at 20 c 20,w (1)

3 Data for the relative viscosity and density of aqueous cesiun chloride solutions were found in Dr. Larre Egbert's procedures book. nrel CsCl d.35m CsCl = 0.982 = 1. 043g cm - 3 Data for the relative viscosity of aqueous solutions of urea were obtained in the International Critical Tables 4, and were plotted to obtain intenrediate points (Fig. 1). An equation for the determination of urea solution density was also found: d = rl + 2.702 x 10-3 (P ) + 3.712 x 10-6 (P ) 2 + -2.285 x 10-8 (P ) 3 'N s s s where P =weight percent of the solution relative to water. (2) 5 s Estimations of the relative viscosities and the densities of the cesium chloride--urea solutions are based on the assunption that these two physical properties are additive---that the addition of urea does not alter the viscosity or density due to cesiun chloride, but does increase the total viscosity and density of the gradient solution. Viscosities for each solution are now calculated by means of: (3) where (nr - 1) is the viscosity contribution of tfue urea. Simiurea larly, the density was calculated: d = d + (d - d,.t ) soln CsCl urea ~O where (d - ~ ) 0 is the density contribution of added urea. The urea ~ 6 density of water at 20 c is.9982 g cm- 3 Additionally, the specific volume 3 -l of the virus is estimated "to be.74 cm g, an average value for viruses.

4 Below is listed the physical values used to calculate s20,w for each urea concentration: gem -3 gem -3 s nr - 1 nr d -c\r d soln urea concentration s 20,w urea urea soln 2 o 0 74 83 0.982 0 1.043 0 73 83 0.982 0 1.043.1 70 80.002.984.002 1. 045.2 72 82.008.990.003 1.046. 2 73 83.008.990.003 1.046.25 59 68.010.992.004 1.047. 25 73 84.010.992.004 1.047.50 72 85.. 022 1.004.009 1.052.5 59 70.022 1.004.009 1.052.7 61 75.028 1.010.012 1.055.9 59 70. 036 1.018.016 1.059 1.0 57 65.039 1.021.017 1.060 1.8 51 69.078 1.060.033 1.076 2.8 43 67.133 1.115.054 1.097 3.7 35 62.193 1.175.075 1.118 s 20,w vs urea concentration has been graphed in figure 2.

Conclusion 5 Examination of the graph shows a general decrease in s 20,w fran about 83s to about 65s, but no concrete conclusions may be drawn. One could say that there is an apparent change in configuration of the virus as indicated by the steep drop in s 20,w between.20 and.25m urea (or between.50 and 1. OM urea depending on the points chosen). However, the number of approx- ' imations necessary to make the conversion between S and s 20,w make it difficult to conclude that a definite change in the virus structure has occurred in a particular urea concentration range. Had there been no configuration change with addition of urea, a horizontal graph \VOUld have been obtained, however--the virus is apparently being changed by the urea. Without further experimentation, a definite conclusion is not possible. Repeated runs are needed to more accurately ascertain values in the.20 to l. OM urea concentration span; duplicate results \\Quld eliminate the indeterminancy. Errors due to viscosity density approximations may be minimized or eliminated by making direct measureiibnts of the gradient solution properties. Corrections to s 20,w using these values should have greater accuracy than those obtained from the approximations. Additionally, experiments with the curly top virus monomer under the same experimental conditions would allow direct comparison of S values without making corrections to s 2 o,w If the dimer separated into monomers, we would expect that the S values would be the same had the monomeric form been added initially. The possibility exists that the rronomeric unit may also be affected by urea, but we \VOuld expect that all of the monomers, regardless of source, would be changed similarly. Examination of the actual configurations of the viruses may be done through electron microscopy, a thj_rd possible area of continued research on this topic.

6 Bibliography 1. Sedimentation Velocity Studies, Larre N. Egbert. SV 129 to SV 235. (unpublished). 2. Vinograd et al., DNAS 49, 902 (1963). 3. Vinograd et al., BIOPOLYMERS ~' 481-489 (1965) 4. International Critical Tables, Vol. y_, p. 22. 5. Ibid., Vol. III, p. 111. 6. CRC Handbook of Chemistry & Physics, 52nd Ed., pp. F-11.

Urea viscosity vs. concentration Fig. 1 oo 50!--- -. +..., ---+ -. -i---1-- --1 - -+-J.--+- --+- -l. '-ty{ -P ' ~ J. ' u - I:.. '1.t 1--1-00 --+-- J -- J-t--=--+--1---+--+--+. --+~+'+' --!--+-+i' I I,1 i -+---+--+- +-+--+--+----'-'--+t -+---+--+--+--+--+--+--+-_E-ll---1---+--l 050 1.0 2.0 3.0-1 urea concentration (M.l ) 4.0

- "'' : :~ f 8 20,w (Svedbergs) S20 w vs Urea concentration Fig. 2 90 l I I I 11 I I I r+ I I I I I'. l t I I f' ~ ff - I 1 +-+ l f I I I ~ I I I f-1 I r I =r I I +: 1 I I I I I+ l l I ii r+i m1++11 ' i + 1-..j- tr...+-i -j +t--+--'++ ++ m ff ~. +H r - : T I u "'; ' t. - I.r:l + + ' I.. 80 ~ ',;. L". '''' " " I -+- + + t ;' 'I I 1d I I I I I..r I I I """I J..r:1. +<" -c:r -f-q- I I ~ I I I I. II rn.. I I 'l H+ '\1 T H +._t-:-: + ~ -.-J ~:±.. :+~-~. n.j I+ J_ t- ~t - + '++ t'. I -t l_j_+. t '. " r::r +::- ' 1- + i.t... 1 +-i '+f'..1 L l I 60 t, 1 t ;t+j_ t.. :. ; t r I' f +..t. 'ct i ' tt.+-...t ', +.. t I +- +. f- 1-1- ---+-:- ~ T. l + -. l' 1. 1 +-!-+ -Ht ~ f+ T.....~±_~ ~' r I I I I H ; ~. r-t-- J_ I --+--.- ' -. +... +- I --j. t--+-;---t-.. -+---+.' l.+ H HI+ ~fl ll.. 1 ' P: l ±it+.. :lfil_ 'i tl-t',_ 'illl llf h:l ] +'++-H ~ ~ n : - T ~ f ~ ~" 1 30 I --r-t - + -+- ~. -t 1 1 iit l j 1 114...,.. J+. +'. '. +1! i:'f.. m - t±.. + t ~ ; ~ i:-~ + tt 7 '. E-ti t + H :..,.. :1 t t rt~ }r. f Jlr ' l ~J;gJ 1 + L::: 1t ~ 1 + +H1hf 1 J l T I ] T11 fift ti 1 ; I 'l t " ; rt di~ +-..il 20 I I l.: ~ t]1 10.. " - LI,_,... _ f-c--+-.. ttf ++. '--\--- --+--+-: +- + +t +n: n ' '... -- - r ' t! 1, J:tl! t+.j.. + L~ r : +t'. ~,... t-'++.,. 1r il ~ " l I I,_' iti-:-;ji+t t-t-r -+--+'-'--+--".. ++- -:- -'+-...-++- ~..i--;.j +-.... +.. + t. '.- )... '+- I - ft! l$,,, '. :r. '.f; +j +++1 t +-+- I. I I ' it t... t 4. + +m '. l-- I -ti -+-+ r -t tttt -r- -t t.. r tit! n: I + + 1 ;:f :-:-+ I mf1t I ' ~rt ::: 1 ffl l 1 - ~: lu t 11 IP 1 I I I I '! - +~-t. -r--t j I I- t--t t. -t-,1----+--t-- I-'-+~++.,.+. ~T ~-+--t---+--+-t---+--+~t---t--+.,---t--+---'-+-'--1 r+r' "!.;-+ 1!1 '):; 'if+ + 0 'T+ I H.! t h.! 1.0 2.0 3.0 4.0-1 urea concentration (M.l )

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