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Macromolecule dry weight determination with a vacuum balance
Macromolecule
Dry
with
Weight
a Vacuum
Keccived
Srpternber
Determination Balance
29, 1969
Precise measurements of physical propert’ies of macromolecules must be accompanied by precise measurements of the sample weight.. For proteins, the most reliable method for estimating the sample weight is the determination of the dry weight. Often this is considered a last resort because it is tedious and requires :I larger sample t’lian one desires to sacrifice. However, in the determinat’ion of absorbance coefficients and in partial specific volume mcasurement,s (1)) the precision of sample weight tleterminations becomes a limiting factor. The apparatus described here has been developed and used over a period of four years in this laboratory and has provided a reliable and convenient method for determining the dry weights of protein samples in the range of 1 to 10 mg. It has proved to be a VCJ’Y necessary adjunct to the magnetic densimcter (1) uecd in our precise measurements of solution densities. The basic instrument is a Cahn electrobalance,’ model RG, intlicated as A in Figure 1, housed in a vacuum bottle (obtained from the same source) modified in our laboratory. At C is a Ilang-down tube providccl with a 30 nmi opening and :t ground-glass closure similar t,o a small desiccator lid. At I> is a thermocouple” for measuring the pressure inside the balance chamber. X XYUXLII~ stopcock” at, E provides an air inlet and one at F provides a valvcl to the col~l trap joined by a mrtal bellow;;, G, with Bovar metal to glass seals. The entire assembly of Figure 1 is support& on :L rack insulatc~(l from nicchnnic:d vibration by shock
mountx4 At U i* :I lin~ig-~lowt t,utxt tIt:tt. hurrouti~l~ the btirrul) 011 whicli the Pample pan is placccl. A dctnilcd view of this hang-down tube i,i provided 1)~ Figure 2. This tulle hna ;L natcr jwlict that provides for low tc111pcKlture cotlt~ol. In t110 s idc is :t 1~01%n-it,11 8, 3.5 cttt dianietw :tiici :t groluld-glass surface that i:: cool~l. This is important since it, ctisurw n cool surface for :I t,iglit gre:w seal. A4t tlit x miw time the grouncl-glass LL(lesiccatorlid” elosurc can lx: 1~~tno~cv1 cwily to provide rcacl~ :tccw to the samplt~ pan. The t,ottotti aimilnrly has :L water-cooled ground-glass surface with a closure that provitlc:: tungsten wires sealed through the glass for electrical lcacls. (Silicone rubber or cposy seals had a tendency to develop pinhole leaks over periods of several months.) This closure i:: detailed in Figure 3. In this figure, A is intended t,o represent the inside wall of the hang-down tube! B is a cylinder of copper ~creett t,hat is grounded through one of the contacts in the closure. This arrttngement minimizes static charge effect,e. &it C is a cylinder of thin aluttiitiutn sheet io increase the cfficiencp of the heating filanicntJ 1) fashioned front To. 30 nichrome wire and having about 25 ohms rcsistancc. The filatnent~ is supported on 3 strip of mica to I)rcvent changes in the geometry an11 position of the filaments. The voltage across t.hc filament. was mnintainetl :IL about 27 V l>y a vokagc trattsfortnw. The cswct. voltage to be uxeti drpends on the tcsmpcratuw desired nticl tlirb gcotnct~t~~of the element?. SRI
’ Barry uwd
Controls, Burhnnk, C’nlii.. mol~nf. for ~llpporiing vflvittim pump
t?‘pv
s~~lwf~~l
for
w-rbiglll
01 r:11.1,. tyitt’
(;OWHICH
.tSI)
REITHEL
29cm
It was found beneficial to inst,all a parabolic aluminum reflector belo\f t,he filament to increase heat t,ransfer. The temperature sensing prohc. E, is a stainless-steel t,hcrmistor of a ‘(banjo” type, 1”s2 in. diameter (YSI 400 series) connected to a tele-thcrrnonlettlr.” This instrument provides both a temperature readin,u anal a means of controlling the current to the filament so that a reasonably constant temperature can be maintained. The shank of the banjo probe was protected by a ceramic sleeve held in place by glass tape in order to minimize conduction effects. The shank of the probe was fastened to one of the leads in the closure and bent at an appropriate angle. The metal bellows (G, Fig. 1) was sealed through a Kovar seal to a simple trap cooled by dry ice. Between t’he trap and the racuum pump” a T was provided for the at.tachment of a precision valve7 leak. For ease in operation the mass adjustment potentiometer of the elec-
-A
--D
-B
1
542
GOODRICH
Ah-D
REITHEL
of the apparatus was begun and, in our apparatus, reached 10 IL in lo-15 min. A record was made of the change in weight with time. When constant, weight was attained at room temperature the pressure was adjusted to 40,~~ with t,he precision valve leak and the heating begun. It is possible to obtain a record of weight changes despite apparent weight fluctuat.ions which form an envelop instead of a line. When the weight no longer changed, the current to the heater was switched off, the apparatus allowed to come to thermal equilibrium, and the pressure adjusted to that of the atmosphere. The final dry weight was then recorded. The time rcquircd to remove water from protein samples varied with the weight of the sample used and the nature of the sample. As an illustrat,ion, a 5 mg sample of bovine serum albumin in phosphate buffer lost 90% of its water in 3 hr when heated to 90” and became constant in weight in 24 hr. Heating seemed to he necessary to remove the last traces of wat,er that are retained by the sample for days cvcn at pressures of 5 IL. The efficiency of heat transfer from the filament to the pan varies markedly with pressure fluctuations. MoreoT-cr, t,he measuring device measures radiation. Therefore, t,hc temperature of the sample was determined by calibrating with melting point capillaries using vanillin (m.p. 85’) and toluic acid (m.p. 102-4”). The variables affecting such a calihrat,ion were the distance of the filament from the stirrup, the pressure, the voltage across the filament,, and the presence or absencr of a reflector surrounding the filament. The efficiency of transfer drops markedly bclow 50 IL, and 40 ,-dwas chosen empirically as a compromise. In general, protein solutions to be sampled for dry weight detcrminations contained only inorganic buffers. Tris acetate, for example, is completely volatilized at 40 p and 85O, and deposits on t’he water-cooled inner surface of the hang-down tube. Whenever a new buffer was to he used its volat’ilit,y was carefully chccked12 under the conditions to be used. The balance maintains its calibration, with respect to standard weights, for the period of the det,ermination, and it is possible to weigh sample pans (about 30 mg) reproducibly to a microgram. Since most protein samples contain buffer it is necessary to estimate the actual protein weight by difference. Since four weighings are involved, and since errors are .additive, it is considered advisable to use samples containing at least 2 mg of prot’ein to ensure accuracy. I2 In order to avoid contamination of the balance with such substances, a small apparatus has been designed in which the sample is warmed to 100” just below a water-cooled surface at a pressure less than 50~. When no sublimation is visibly, the sample may be transferred to thr vacuum balance. A sketch of t,his accessory will hr srnt on requc,ct.
Dry
with
Weight
a Vacuum
Keccived
Srpternber
Determination Balance
29, 1969
Precise measurements of physical propert’ies of macromolecules must be accompanied by precise measurements of the sample weight.. For proteins, the most reliable method for estimating the sample weight is the determination of the dry weight. Often this is considered a last resort because it is tedious and requires :I larger sample t’lian one desires to sacrifice. However, in the determinat’ion of absorbance coefficients and in partial specific volume mcasurement,s (1)) the precision of sample weight tleterminations becomes a limiting factor. The apparatus described here has been developed and used over a period of four years in this laboratory and has provided a reliable and convenient method for determining the dry weights of protein samples in the range of 1 to 10 mg. It has proved to be a VCJ’Y necessary adjunct to the magnetic densimcter (1) uecd in our precise measurements of solution densities. The basic instrument is a Cahn electrobalance,’ model RG, intlicated as A in Figure 1, housed in a vacuum bottle (obtained from the same source) modified in our laboratory. At C is a Ilang-down tube providccl with a 30 nmi opening and :t ground-glass closure similar t,o a small desiccator lid. At I> is a thermocouple” for measuring the pressure inside the balance chamber. X XYUXLII~ stopcock” at, E provides an air inlet and one at F provides a valvcl to the col~l trap joined by a mrtal bellow;;, G, with Bovar metal to glass seals. The entire assembly of Figure 1 is support& on :L rack insulatc~(l from nicchnnic:d vibration by shock
mountx4 At U i* :I lin~ig-~lowt t,utxt tIt:tt. hurrouti~l~ the btirrul) 011 whicli the Pample pan is placccl. A dctnilcd view of this hang-down tube i,i provided 1)~ Figure 2. This tulle hna ;L natcr jwlict that provides for low tc111pcKlture cotlt~ol. In t110 s idc is :t 1~01%n-it,11 8, 3.5 cttt dianietw :tiici :t groluld-glass surface that i:: cool~l. This is important since it, ctisurw n cool surface for :I t,iglit gre:w seal. A4t tlit x miw time the grouncl-glass LL(lesiccatorlid” elosurc can lx: 1~~tno~cv1 cwily to provide rcacl~ :tccw to the samplt~ pan. The t,ottotti aimilnrly has :L water-cooled ground-glass surface with a closure that provitlc:: tungsten wires sealed through the glass for electrical lcacls. (Silicone rubber or cposy seals had a tendency to develop pinhole leaks over periods of several months.) This closure i:: detailed in Figure 3. In this figure, A is intended t,o represent the inside wall of the hang-down tube! B is a cylinder of copper ~creett t,hat is grounded through one of the contacts in the closure. This arrttngement minimizes static charge effect,e. &it C is a cylinder of thin aluttiitiutn sheet io increase the cfficiencp of the heating filanicntJ 1) fashioned front To. 30 nichrome wire and having about 25 ohms rcsistancc. The filatnent~ is supported on 3 strip of mica to I)rcvent changes in the geometry an11 position of the filaments. The voltage across t.hc filament. was mnintainetl :IL about 27 V l>y a vokagc trattsfortnw. The cswct. voltage to be uxeti drpends on the tcsmpcratuw desired nticl tlirb gcotnct~t~~of the element?. SRI
’ Barry uwd
Controls, Burhnnk, C’nlii.. mol~nf. for ~llpporiing vflvittim pump
t?‘pv
s~~lwf~~l
for
w-rbiglll
01 r:11.1,. tyitt’
(;OWHICH
.tSI)
REITHEL
29cm
It was found beneficial to inst,all a parabolic aluminum reflector belo\f t,he filament to increase heat t,ransfer. The temperature sensing prohc. E, is a stainless-steel t,hcrmistor of a ‘(banjo” type, 1”s2 in. diameter (YSI 400 series) connected to a tele-thcrrnonlettlr.” This instrument provides both a temperature readin,u anal a means of controlling the current to the filament so that a reasonably constant temperature can be maintained. The shank of the banjo probe was protected by a ceramic sleeve held in place by glass tape in order to minimize conduction effects. The shank of the probe was fastened to one of the leads in the closure and bent at an appropriate angle. The metal bellows (G, Fig. 1) was sealed through a Kovar seal to a simple trap cooled by dry ice. Between t’he trap and the racuum pump” a T was provided for the at.tachment of a precision valve7 leak. For ease in operation the mass adjustment potentiometer of the elec-
-A
--D
-B
1
542
GOODRICH
Ah-D
REITHEL
of the apparatus was begun and, in our apparatus, reached 10 IL in lo-15 min. A record was made of the change in weight with time. When constant, weight was attained at room temperature the pressure was adjusted to 40,~~ with t,he precision valve leak and the heating begun. It is possible to obtain a record of weight changes despite apparent weight fluctuat.ions which form an envelop instead of a line. When the weight no longer changed, the current to the heater was switched off, the apparatus allowed to come to thermal equilibrium, and the pressure adjusted to that of the atmosphere. The final dry weight was then recorded. The time rcquircd to remove water from protein samples varied with the weight of the sample used and the nature of the sample. As an illustrat,ion, a 5 mg sample of bovine serum albumin in phosphate buffer lost 90% of its water in 3 hr when heated to 90” and became constant in weight in 24 hr. Heating seemed to he necessary to remove the last traces of wat,er that are retained by the sample for days cvcn at pressures of 5 IL. The efficiency of heat transfer from the filament to the pan varies markedly with pressure fluctuations. MoreoT-cr, t,he measuring device measures radiation. Therefore, t,hc temperature of the sample was determined by calibrating with melting point capillaries using vanillin (m.p. 85’) and toluic acid (m.p. 102-4”). The variables affecting such a calihrat,ion were the distance of the filament from the stirrup, the pressure, the voltage across the filament,, and the presence or absencr of a reflector surrounding the filament. The efficiency of transfer drops markedly bclow 50 IL, and 40 ,-dwas chosen empirically as a compromise. In general, protein solutions to be sampled for dry weight detcrminations contained only inorganic buffers. Tris acetate, for example, is completely volatilized at 40 p and 85O, and deposits on t’he water-cooled inner surface of the hang-down tube. Whenever a new buffer was to he used its volat’ilit,y was carefully chccked12 under the conditions to be used. The balance maintains its calibration, with respect to standard weights, for the period of the det,ermination, and it is possible to weigh sample pans (about 30 mg) reproducibly to a microgram. Since most protein samples contain buffer it is necessary to estimate the actual protein weight by difference. Since four weighings are involved, and since errors are .additive, it is considered advisable to use samples containing at least 2 mg of prot’ein to ensure accuracy. I2 In order to avoid contamination of the balance with such substances, a small apparatus has been designed in which the sample is warmed to 100” just below a water-cooled surface at a pressure less than 50~. When no sublimation is visibly, the sample may be transferred to thr vacuum balance. A sketch of t,his accessory will hr srnt on requc,ct.