Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate

ANALYTICAI

68, 358-370

HIOCHEMISTRY

Electrophoretic Sulfate

Behavior

Complexes

Amounts

This sulfate

report present

tein-dodecyl mational these spectra

describes

the

solution

structural changes and and circular dichroism of the

same

amounts

forming enhanced teins that

a separation.

of Various

Dodecyl

between and

the

the

Sulfate

amount

of sodium

electrophoretic

mobility

In order to determine the extent and to establish a correlation the electrophoretic spectra were of surfactants

it is apparent solution must

Optimum

separation and can be accurately

Dodecyl

in the Presence

relationship

in a sample

From the results obtained. fate present in the sample

of Protein

of Sodium

sulfate complexes. changes in the proteins

presence

( 1975)

behavior, for

as used that the he taken

experimental

a maximized determined.

obtained

range

of any between

pro-

conforany of

visible absorption proteins in the

in electrophoresis.

amount of sodium into consideration

conditions linear

heme

dodecyl of the

are of molecular

chosen

dodecyl when for weights

sulper-

attaining of pro-

A variety of proteins have been separated and their molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis. Since it is an extensively used technique, procedures have been employed to determine its reliability in terms of the charge of the native protein (l-3). the conformation of the native protein (3,4) and the range of protein molecular weights that can be accurately determined (5-9). Several of the experimental conditions that have been varied include acrylamide concentration (5,6.8). amount of cross-linker (5,6), addition of urea to the system (6,8,lO), amount of a reducing agent such as /X-mercaptoethanol (I I), amount of N, N, N’, N’-tetramethylethylenediamine (12) and the use of disk electrophoresis (9). Little information has been reported on the effect of the amount of SDS present ( 13). Shapiro et nl. first observed that the relative mobilities (RM) of various proteins are dependent on their molecular weights in the presence of SDS ( 13). This dependency on molecular weight is based on the ’ Address

correspondence

to this

author-

at Ohio

358 Copyright All rights

i-1 lY75 by Academic Press, Inc. of reproduction in any form reserved.

University

ELECTROPHORETIC

MOBILITY

OF

PROTEIN-SDS

359

fact that DS binds to protein5 aon a gram to gram ratio ( 14.15). Therefore the charge to mass ratio ((>/,?I) of these proteins is the same at a specific binding ratio, and thus their migration in a polyacrylamide-gel medium under the influence of an electric field depends on the length of the protein complex chain which is a function of the molecular weight of the protein ( 16). Assuming there are different binding ratios, it would seem that the RM would be affected due to the influence of the binding ratio on (J/W. Besides the effect on c~/nz, SDS can also alter the structure of the protein. which is another important factor that controls the mobility of a protein-DS complex. Therefore the extent to which various amounts of SDS alter the structure must be considered. Information concerning any conformational changes can be obtained by observing the spectral properties of protein-DS complexes with absorption spectrophotometry and circular dichroism. Studies have been done concerning the effect on the visible absorption of heme proteins in certain denaturants such a:, SDS. urea and organic solvents ( 17-30). These spectral changes are attributed to the denaturation process. Circular dichroism has been trsed to determine the effect of these denaturing agents on the secondary structure ot proteins (2%76). The purpose of this investigation was to obtain various binding ratios of SDS to protein under constant experimental conditions and determine the effects of these ratios on the /<1%2’s and spectral properties of various proteins. The procedure employed was based on an earlier publication where it was concluded that SDS is needed just in the sample solution applied to the top of each gel prior to electrophoresis (37). The results indicated that the proteins were saturated at high ratios of SDS to protein and therefore it was not necessary to include SDS both in buffer compartments and in the gels. Also this procedure did not include heating of the sample or the addition of urea because it was observed that these conditions did not alter the KM’s when high ratios of SDS to protein in the sample solution applied to each gel were used. In order to maintain constant experimental conditions for this investigation of the effects of various binding ratios. heating and urea addition were not employed for any ratio of SDS to protein. MATERIALS NaH,PO,.H,O. Na,HPO,.7H,O. ammonium persulfate (AP) and bromophenol blue were purchased from Fischer Scientific Co. Cyanogum 41 (5% N.N-methylene-bisacrylamide and 955% acrylamide) was obtained from Nutritional Biochemicals Corp. SDS of a highly pure grade was obtained from Pierce Chemical Co. Coomassie brilliant blue R 20 was supplied by Sigma Chemical Co. N,N.N’,N’-tetramethyIethylenediamine (TEMED) was a product of Bio-Rad Laboratories.

360

STOKLOSA

AND

TABLE P~ortrw

USED

I IN SIX

Serum

albumin

trimel

Serum Serum

albumin albumin

dimcl monomer

Catalase y-Glohulin(H Egg alhnmin

chain)

Pqxin ~u-C’hyillotryp~inojien y-GlohulintL chain)

A

Ly\orymc Rihonucleasc chain) (’ chain)

Indin

” Sigma

Biochemicals Chemical

weight

Source

204 -000 I36.000 68 .ooo

N.B.C.” N.B.C. N.B.C.

60.000 55.000

S.C.C.” N.B.C.

43.000 35.000

S.C.C. S.C.C. S.C.C.

25.700 23.500 17.100

Ilyoglobin Hemogiohin

” Nutritional

STLTD~

Molecular

Protein

Chymotrypsin(C Cytochrome Chym~,trypsin(B

LATZ

N.B.C.

I5 200 14.400

S.C.C. S.C.C. S.C.C.

13.700 13.000 I I.700

S.C.C. S.C.C. S.C.C.

1 I .ooo 5.700

S.C.C. S.C.C.

Corp

Co.

All other chemicals in Table l.

were

reagent

grade. The proteins

used are listed

METHODS El~,c.tl.ollllo~c,\.i,~. The procedure was based on the one described by Weber and Osborn (5) with the modification of using SDS in the sample solution only (27). Experimental conditions such as gel concentration, applied current, time of run etc. were held constant with the only variable being the SDS in the sample solution applied to the top of each gel prior to electrophoretic separation. The buffer used in the electrode vessels and in the gels was 0.1 M phosphate at pH 7.2. To insure that no SDS was present in the system except for that which was in the sample solution. fresh buffer was used for each run. All the gels were formed from a solution of 5% Cyanogum, 0.15% TEMED and 0. I% AP. The SDS solutions used to prepare 0.5 mg/ml protein solutions contained the desired percentage of SDS (0-0.3V~), 0.1% P-mercaptoethanol and 0.01 M phosphate at pH 7.3. Since the composition of the sample solution applied to the top of each gel prior to electrophoretic separation determined the electrophoretic mobility of the protein-DS complexes, a standard procedure was used. This procedure was as follows: 20 ~1 of the protein solution which had

ELECTROPHORETIC

MOBILITY

OF

PROTEIN-SDS

361

been prepared in the presence of SDS 11-15 hr before use. 25 ~1 of the appropriate SDS solution which had been used to dissolve the proteins. 5 ~1 of fi-mercaptoethanol, I5 PI of 0.3% bromophenol blue which was used as the marker dye and one drop of glycerol were mixed together and applied to the top of each gel. A constant current of 6 mA per tube was applied for 3.5-to 4 hr. Staining and destaining were undertaken in the manner described by Weber and Osborn (5). An additional study was performed that involved the variation of the standard procedure previously described for preparation of sample solutions prior to electrophoresis. The proteins used in this study were myoglobin and hemoglobin. Each procedure employed was based on the standard procedure except that one or more of the conditions for sample preparation were altered. These conditions are as follows: A) No additional 32 ~1 of the appropriate SDS solution was used. B) Addition of 2 ~1 of a solution containing just 0.01 M phosphate at pH 7.2 and 0.1% /3mercaptoethanol. C) Addition of 50 ,u*I of the SDS solution. D) Proteins were dissolved in 0.01 M phosphate at pH 7.2 and 0.17~ /3-mercaptoethanol with no SDS present. As the samples were prepared for their application to the gels prior to a run. 25 ~1 of the SDS solution was added. E) Same as procedure D except that 50 PI of the SDS solution was added. F) Proteins were dissolved in the SDS solution but their concentration was reduced to 0.25 mg/ml. No additional 2.5 ,ul of the SDS solution was used. G) Same as procedure F except 23 ~1 of the SDS solution was added. The ratio of micrograms of SDS to micrograms of protein present at the time of application of the sample solution to each gel was calculated by determining the total SDS present which was the sum of the SDS in the 20 ~1 of protein solution pt-epared I?- I5 hr before use and any other SDS added prior to a run. This sum was divided by the amount of protein applied to the top of each gel. These ratios are not intended to imply that all DS was bound to the protein. l,‘isihlc ~lhsorptio~l .cpc~c’tlol~llotot?l~~t~.~. Spectral measurements were made with a Cary I4 recording spectrophotometer. The region from 300-430 nm was scanned for catalase. myoglobin. hemoglobin and cytochrome (‘. The solutions for this study were prepared under the same conditions as those used in the standard procedure for electrophoresis except that no glycerol. bromophenol blue or fi-mercaptoethanol were used. and of course larger volumes of these solutions were required. Circ,///rrr. tlic~lrroisrrr. Circular dichroism measurements were performed with a Jasco J-20 automatic recording spectra-polarimeter. The region from 180-200 nm was scanned for catalase. myoglobin. hemoglobin and cytochrome c. The protein solutions were pi-epared in the exact same manner as those for the visible absorption study.

362

STOKLOSA

AND

LATZ

RESULTS For each protein an average KM was calculated, based on a minimum of 19 separate runs, at various SDS levels with all the other experimental conditions constant. Depending on the protein, a change in the amount of SDS present produces a corresponding change in the RM. A graphical representation of this relationship between RM and SDS/protein ratio for serum albumin pepsin, myoglobin and hemoglobin is shown in Fig. 1. Proteins exhibiting these variations in RM have ranges of SDS/protein (weight ratio) where their respective RM’s are affected in a manner similar to that illustrated in Table 3. For ratios of 1.35-4.50 there is a linear increase in RM for most of the proteins with molecular weights of 43,000 or less. Beyond the 4.50 ratio there is only a slight increase in RM with an increase in the ratio of SDS to protein. However the extent of this increase depends on the molecular weight of the particular protein. From O-0.90 there are generally no bands at all, or, if there are, they are diffuse with a RM less than that predicted from the linear relationship observed for ratios ranging from 1.35-4.50. For the higher molecular weight proteins, a maximum RM is observed at a ratio of 1.35 with no significant change in RM beyond this point. In regard to certain characteristics of the observable bands, such as width, intensity and sharpness, there appears to be no change from a

1.0

I

gt

6

41 0

1.0

2.0

I

30

40

50

60

Mtcrograms

FIG.

myoglobin constant

I. The

effect

on

the

(a) and hemoglobin amount of protein.

of

electrophoretic (0)

70 SDS

80

per

m~crogrom

migration

in the presence

90

100 of

110

130

140

150

pro+e~n

of serum of various

120

albumin

amounts

(0).

pepsin

(LJ).

of SDS

relative

to a

ELECTROPHORETIC

Er~rcr

OF

ri~r.

MOBILITY

.~~IOCNT MOBILITY

TABLE OF SDS OF SEVERAL

OF

2 PRFS~N

363

PROTEIN-SDS

I oix

THI:

RF.L.AY IVF

PRWFINS KM

Protein Serum Serum Serum

albumin albumin albumin

Cntalase y-Globulin

trimet dimet monomer

(H

chain)

Egg Albumin Pepsin wChymotrypsinogen y-Globulin tL chain) Myoglobin Hemoglobin Lywzymc Rihonucleaxe Chymotrypsin Cytochrome Chymorrypsin Insulin

(B chain) (’ (C chain)

.4

0-0.90

I .i-4.50

5.40-13.5

04.27 0.35-0.3s 0.37-0.58

0.1X-0.2X 0.36-0.36 0.62-0.64

0.27-0.26 0.36-0.37 0.64-0.62

0.26-0.67 O-O

0.67-0.67 O-O.66

0.66-0.65 0.66-0.66

0.51-0.71 0.60-0.71 O-O o-o.75 O-O.79

0.74-0.77 0.76-0.81 o-o. 8X 0.7Y-0.91 0.x71 .I12

0.76-0.77 0.X5-0.86 O.YM~.XX 0.91-0.90 1.04- 1.07

0-0.x1 o-0.74 o-o .70

o.xx- I .os O.XI-I.OI 0.7%0.98

1.06-1.11 1.01-1.08 I .oo- I .Ol

04.80 O-0.69 0-0.x0

0.X% 0.x30.x5-

1.05-1.12 1.01-1.06

0.24-0.77

0.X8-1.06

I .O? I .oo I .02

I .O5- I. Ii I .07-I. I6

1.35-l 3.50 ratio. As stated previously at least 12 separate runs were made for each protein at a specific ratio. The calculated KM’s have relative average deviations of less than 35%. As shown in Fig. I. there is only a slight linear increase in RM for pepsin from a 1.35-4.50 ratio. However for myoglobin and hemoglobin there is a greater increase in RM. By plotting the slopes of this lineat portion against the molecular weights of the proteins, there appears to be a linear dependency of this change in RM on the molecular weight of the protein. This relationship is illustrated in Fig. 2. In order to determine the importance of several of the other conditions employed in preparing the sample which was applied to the top of each gel prior to electrophoresis, different procedures of obtaining the same ratio of SDS to protein as that obtained with the standard procedure were used. A comparison of the RM's with these different procedures and those with the standard procedure is given in Table 3. Procedure A involved the elimination of the additional 25 ~1 of the SDS solution prior to a run. Based on just the SDS present in the original 20 ~1 of the protein solution, the resulting KM's compare favorably with those predicted from Fig. I. The addition of 75 ~1 of a solution con-

364

STOKLOSA

I IO

1 0

AND

I

I 30

20 M. w.

FIG. tein-DS Ratio)

2. The dependency complex on the were

calculated

SDS to protein from the protein complexes.

LATZ

of the molecular

from

the

a I .35-4.50

ratio.

I 50

x 10-3

rate of increase weight of the

lineal-

I 40

in the protein.

portion

in the

This

relationship

relative The

relationship

mobility (KM) rates of increase between

is illustrated

RM in Fig.

of a pro(1 /
ratio

of

I for

four

of

taining 0.01 M phosphate at pH 7.2 and 0.1% ,?kmercaptoethanol with no SDS present, which would not affect the ratio of SDS to protein, does not appear to affect the RM. Since it is apparent that the additional 7-5 ~1 of the SDS solution does affect the RM, the volume of this SDS solution added prior to a run was increased to 50 ~1. This results in the protein complexes having a higher RM than those obtained when 25 ~1 of the SDS solution was used. To illustrate further the importance of this SDS solution added prior to electrophoretic separation. the proteins were dissolved in a solution containing 0.01 M phosphate at pH 7.2, 0.1% p-mercaptoethanol and no SDS. Prior to the electrophoretic separation, SDS was added. Intense sharp bands were observed with RM's similar to those predicted from the calculated ratios. Using this same procedure of not including any SDS until just before a run was made. bands were observed for all the proteins listed in Table I except for yglobulin (H and L chains) and a-chymotrypsinogen A. A calibration curve of log M,. vs RM was constructed, and the molecular weight of malate dehydrogenase, isolated by E. M. Gregory,” was verified even L Private

consultation

in this

laboratory.

ELECTROPHORETIC

~‘HL

C~~IPARIS~N

MOBILITY

OF Rtl PROCEDLIRC Ratio

:ATIV~ WITH

OF

M~HILITIES MODIFIEI)

0.30 0.20 0.IO 0.30 C).20 0.30 0.20 0.30 0.20 0.30 0.20 0.30 0.20 0.10 0.0 0.30 0.IO O.OF

H c I> E F

G

” Mh.

myoglohin:

Hh.

OB.IAINFD PROCEDL’RFS

~‘r

SIA~VDAKD

of SDS

R,tf,,,,

to protein

.4

365

PROTEIN-SDS

I .w

I.00 0.90

I .06 1 .ni n.91

1.07 I .07 0.91

I .05 I .no

I .07 I .03

I .07 I .oi

I .0x I .0x

1 12 I.12

I .Oh I .04

I .0x I .06

I .0x

I .07

I.11 I .0x

I.17 I .09

I .08

I.10

0.94 O.YO

l.0h 0.96 O.Yl

I.08 I .03 O.Y2

I .07 I .n5 0.9?

1.09 I.08 0.w

I .06 I .M I .04

I .06

-, I.11 I .OY I .05

hemoglobin.

though the initial protein solution contained no SDS and only prior to electrophoresis (- IS min) was SDS used. The final condition that was varied was the concentration of the proteins. As shown in Table 3 for procedures F and G, the observed KM's were generally less than those predicted from the calculated ratios of SDS to protein. The presence of various amounts of SDS produces changes in both the wavelengths of maximum absorption (h,,,;,,) and the absorbances (A) for the catalase. myoglohin. hemoglobin and cytochrome c in the region from 400-4-30 nm. For catalase. myoglobin and hemoglobin there is an increase in h,,, and a decrease in rl from 0 to a 0.45 ratio. (‘ytochrome (’ exhibits a decrease in A,,;,, and an increase in .+I over this range of ratios. There is little or no change in these values from a 0.45-3.60 ratio except for catalase that begins to show a decrease in A,,, and rl at a 3.60 ratio. From a 4.50-45.0 ratio there is a decrease in X,,, and an initial decrease in A with no significant change beyond this point. These efin Table 4. fects on A,,,;,, and /I are summarized

366

STOKLOSA

EFFECT

OF ‘THE AMOUNT WAVELENGTH

AND

LATZ

TABLE 4 OF SDS ON THE ABSORBANCES MAXIMA OF Ht~t PROTEINS

Micrograms

of SDS per microgram

Catalase Myoglobin Hemoglobin Cytochrome

A

&X,,

c

402-410 40X-4 I4 406-4 I3 411-40X

0.35-0.32 1.74-1.36 1.56-1.27 1.33-1.47

THE

of protein

0.90-3.60

O-O.45 Protein

AND

4.50-45.0

A,,,;,,

A

4 I O-404 414-414 413-412 40X-408

0.24-O. 19 1.35-1.x 1.17-1.10 1.56-1.51

A

h,, 404-400 41 l-402 40X-406 406-40 I

0. IX-O. 18 0.91-0.91 0.X4-0.90 I .36-l .23

Since it was observed that intense sharp bands were obtained for myoglobin and hemoglobin following electrophoretic separation even though these proteins were not dissolved in an SDS solution overnight and only came in contact with SDS prior to a run, the absorbance of myoglobin was recorded for each ratio of SDS to protein within approximately 3 min after mixing the protein solution containing no SDS and the SDS solution. These absorbances are the same as those previously obtained with the standard procedure where myoglobin was dissolved in the SDS solution 12-1.5 hr prior to their use. The circular dichroism spectra of catalase, myoglobin, hemoglobin and cytochrome c consist of two negative maxima in the region from 200-280 nm which is characteristic of protein exhibiting a-helical strucTABLE 5 OF THE AMouwr OF SDS ON THE MOLAR ELLIPTICITIES OF HEME PROTEINS

EFFECT

Micrograms o-o.45 Protein

A lli‘il

of SDS per microgram

of protein

0.90-3.60 [H] x IOV

A,,,;,,

4.50-45.0

[H] x lo+

~,,,a,

[H] x 10-f’

Catalase

218-218 108-208

4.35-5.34 4.51-6.54

21X-218 206-206

6.35-6.35 8.73-X.72

71X-218 206-206

6.35-6.35 8.6X-6.71

Myoglobin

190-2 I8 30X-208

16.6-l 1.9 15.3-13.7

2x-270 20X-208

13.2-13.2 14.3-14.3

21X-218 ‘07-107

13.2-13.2 1.5-15.5

Hemoglobin

290-220 10X-208

11.6-11.2 13.6-12.4

220-220 207-207

12.4-12.4 14.3-14.4

220-220 20X-208

13.0-13.0 14.4-14.4

2’1-221 90X-707

6.84-6.58 5.44-7.3x

220-270 206-206

7.11-7.11 9.26-9.26

220-210 ZOh-‘Oh

7.3X-7.38 10.6-10.6

Cytochrome

<’

ELECTROPHORETIC

MOBILITY

OF

PROTEIN-SDS

367

cjf the molar ellipticitiec [H] at these ture. Changes in the magnitude maxima were observed over three ranges of ratios of SDS to protein as illustrated in Table 5. For myoglobin, hemoglobin and cytochromc tl there is a slight decrease in [H] from 0 to a 0.45 ratio. Catalase has an increase in [H] over this range. For all four proteins there is an increase in [H] at a 0.90 ratio with no change in this [H] up to 3.60. At a 4.50 ratio there is another increase in [H] for myoglobin, hemoglobin and cytochrome (‘. Beyond this point [H] remains constant. Catalase has a constant IN] from a 0.90-45 ratio. DISCUSSION The results of this investigation indicate that the amount of SDS present does have a decided effect on the KM of the protein-DS complexes. In order to determine the possible reasons for this apparent dependency of the RM on the amount of SDS present, the basic principles involved with gel electrophoresis must be considered. A theoretical treatment of the parameters governing the electrophoretic migration of protein-DS complexes has been outlined by Rodbard and Chrambach (38). Two important parameters involved are the apparent free mobility (Y,,) and the retardation coefficient (K,,). The relationship between these two parameters and the obrerved RM is given in the following equation first proposed by Ferguson (30):

where 7‘ is the total monomer concentration. K,, is a function of molecular size and the percent of cross-linking and Y,, is the mobility at T = 0. Based on this equation, the RM depends on just K,, as long as T and l’,, remain constant. The Y,, value has to be the same for each protein-DS over the entire range of molecular weights. Under these conditions RM varies with the molecular weight of the protein since the length of the denatured protein complex chain is dependent on the molecular weight ( 16). For Y,, to remain constant and independent of RM and KE,, the various protein-DS complexes must behave as if they are under free electrophoresis conditions (T = 0). This means that the RM of both large molecular weight proteins (serum albumin trimer) and small proteins (hemoglobin) are the same at T = 0. Several workers have reported Y,, values of various protein-DS complexes (8, 9, 29). Their results. which were obtained by recording the RM at various gel concentrations (7‘) and then extrapolating to 7‘ = 0, indicate that Y,, was nearly the same value for a large number of protein complexes. This is due to the fact that the e/r?z ratio was constant and this value is maintained by a specific binding ratio of DS to all the various proteins. The small variations in Y,, they observed are attributed to structural effects of the complexes.

368

STOKLOSA

0

AND

LATZ

I

1

1

1

I

I

2

4

.6

.8

1.0

1.2

R.M. FIG. lar

3. The

weights

extent of

various

of linearity proteins

in the

relationship

at a I.35

CA)

between and

a 13.5

the

logarithms

tC’)

ratio

of

of the SDS

to

molecuprotein.

At small ratios of SDS to protein, the migration of each protein complex is a function of an increase in its l~/nz and a change in its structure, both of which are caused by an increase in the binding ratio of DS to protein. Over an intermediate range of ratios of SDS to protein, the migration of each complex is a function of an increase in its o/t~z with its structure being unaltered by an increase in the binding ratio. At higher ratios of SDS to protein where the increase in KM no longer adheres to the linear increase observed with the intermediate ratios of SDS to protein. the deviation from linearity is due to micelle formation which can compete with protein-DS formation, structural transformation and the approach of saturation of the protein-DS complex. Since the KM’s of the protein-DS complexes depend on the ratio of SDS to protein in the sample solution, a high ratio is used in order to obtain the most effective method for electrophoretic molecular weight

ELECTROPHORETIC

MOBILITY

OF

369

PROTEIN-SDS

determinations. At this ratio the linear range of molecular weights observed in a log M,. vs RM plot (Fig. 3) and the separation factor are at a maximum. Since the variation in the RM’s of the protein complexes is minimal at the higher ratios. a greater error in the amount of SDS used in the sample solutions and the protein solutions is tolerated. y-Globulin (H chain] and cu-chymotrypsinogen A have no observable bands until higher ratios are used. Possibly other proteins would act in a similar manner. Also it is advantageous to use high ratios when small amounts of protein are used since the RM’s of a protein complex at high ratios are similar regardless of the amount of protein. In terms of structural stability, there are no alterations in the protein complex conformation at high ratios. However even at these high ratios the amount of SDS necessary for a complete analysis is t-educed by a factor of at least 500 in comparison with a procedure using 0.1% SDS in the 50%ml uppet buffer compartment and gels. In regard to the stability of the protein complexes formed at each of the various ratios as a function of the time that the constant current was applied. the RM’s of the complexes at high and low ratios were not dependent on this time of electrophoresis. This indicated that little or no bound DS was lost with time. ACKNOWLEDGMENTS WC

thank

tit-cular

Vaugh

dichroi\m

Vandegrift

and

Martin

Serra

fol-

their

a\\i\tance

in

obtaining

the

i‘~~i~l/~l!i,l.

42.

~pcctra.

REFERENCES I. Tung. J. 1117-1121. 2. ?.

Panyim. Dunker.

S..

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I:~,!~ilx.\.

246, 7557-7560. C‘lrc,,?~. 144, 5073-5080.

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X.

C.

(‘.

Banket-.

Y. Neville. IO. Ghan. I I. I?. Ii.

A..

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<‘orman.

W. t IY72)

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Richter-Landjherg. C.. Ruchel. RCA. C‘c,tlr,rr/,/r. 59. 78 I -7X8. Allison. J. H.. Agrawal. H. C..

K.. and

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Moore.

14.

Reynolds.

J. A..

and

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J. A., T.

and T..

4i44-4550.

Tanford. Tanford. .laillet.

c‘.

( 1970)

Prr~.

C‘. ( I Y70) .I. Uirjl. H.. and Gadrgheku.

247.

F. V.

B. W.

IF.

39. 462-477. 57, 11 I - 12.5. 5X56-5Xh

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