New Applications of Lasers to Chemistry - ACS Publications

7 The Study of Biological Surfaces by Laser

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Electrophoretic Light Scattering B. R. WARE Department of Chemistry, Harvard University, 12 Oxford Street, Cambridge, MA 02138

One of the newest a p p l i c a t i o n s of l a s e r s is the measurement of very small v e l o c i t i e s through the Doppler e f f e c t on l a s e r light which has been s c a t t e r e d or r e f l e c t e d from moving o b j e c t s . This technique, c a l l e d l a s e r Doppler velocimetry, has been a p p l i e d t o a number of i n t e r e s t i n g biological problems, and a general review of the progress i n t h i s area has r e c e n t l y been p u b l i s h e d (1). This l e c t u r e will focus on the a p p l i c a t i o n of l a s e r Doppler velocimetry t o the d e t e c t i o n of e l e c t r o p h o r e s i s , a technique which I s h a l l call e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g (ELS). The theory and the first s u c c e s s f u l experiments of t h i s type were reported by myself and Bill Flygare i n 1971 (2). Since then the technique has been developed and a p p l i e d by s e v e r a l groups t o a wide v a r i e t y of problems. For reviews see references (1/3/4/5). I shall s t a t e briefly the p r i n c i p l e s of e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g , describe the methodology of the experiments as we do them, and then summarize the r e s u l t s of a few of the p r o j e c t s which are being pursued in my l a b o r a t o r i e s at Harvard. A block diagram of an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g apparatus i s shown i n Figure 1. Laser l i g h t i l l u m i n a t e s the p a r t i c l e s or molecules t o be s t u d i e d , and l i g h t which has been s c a t t e r e d from these p a r t i c l e s at a s e l e c t e d angle θ i s c o l l e c t e d and d i r e c t e d t o a p h o t o m u l t i p l i e r tube. When an e l e c t r i c f i e l d i s a p p l i e d to the suspension, the p a r t i c l e s migrate toward the e l e c t r o d e of opposite p o l a r i t y . L i g h t s c a t t e r e d from them i s t h e r e f o r e s l i g h t l y s h i f t e d i n frequency by the Doppler e f f e c t . In order t o measure t h i s s h i f t , i t i s necessary t o combine with the s c a t t e r e d l i g h t a beam of u n s h i f t e d l i g h t which i s obtained by s p l i t t i n g a second beam from the i n c i d e n t l a s e r l i g h t and bypassing the chamber. These two beams, the s c a t t e r e d l i g h t and the s o - c a l l e d l o c a l o s c i l l a t o r beam, are mixed at the photocathode t o produce a low-frequency beat which i s e x a c t l y the Doppler s h i f t frequency. The low-frequency o s c i l l a t i o n s of the photocurrent are then a m p l i f i e d and processed by a real-time spectrum analyzer, the output of which i s the complete spectrum 0-8412-0459-4/78/47-085-102$05.00/0

© 1978 American Chemical Society

Hieftje; New Applications of Lasers to Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Laser Electrophoretic Light Scattering

VARIABLE SLIT VIEWING SCREEN VARIABLE ATTENUATOR

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Contemporary Topics in Analytical and Clinical Chemistry

Figure 1.

Diagram of an electrophoretic light-scattering apparatus.

The coherent, monochromatic beam from the laser is split into two beams. The forward beam is focused into the electrophoretic light-scattering chamber to illumhMe the moving particles. The chamber sits at an angle to the beam in order to obtain a higher scattering angle Θ, so the beam is refracted on entrance and exit. A constant electric field is applied to the scattering region by applying pulses of constant current to the electrodes. Duration of and interval between pulses are controlled by a specially constructed timing circuit. Light is scattered from the particles which are drifting in the electric field, and the scattered light is therefore shifted slightly in frequency by the Doppler effect. The scattered light is collected by an optical system and focused onto the surface of a photodetector. The split-off beam is recombined with the scattered light at the window of the chamber in order to form the so-called local oscillator. The alignment of the scattered light and the local oscilhtor is facilitated by the formation of real images of both, which are mewed on a screen when deflected by a reflex mirror. Once the two have been aligned at the proper point on the screen correspond ing to the center of the photocathode, the reflex mirror is moved out of the way, and the two beams pass through a calibrated slit to the photocathode. There they produce a beat or spectrum of beats equal to the Doppler shift magnitudes between the shifted scattered light and the unshifted local oscillator. The photocurrent or voltage is then amplified and analyzed in frequency by a real-time spectrum analyzer, which is triggered by the timing circuit to accept data only when the field is on. The diagram of the scattering chamber is shown greatly enlarged and is highly schematic. For actual designs and descriptions, see Ref. 5.


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o f a l l Doppler s i g n a l s corresponding t o the histogram of v e l o c i t i e s i n the sample. The equipment r e q u i r e d f o r an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g experiment i s r e l a t i v e l y simply and inexpensive. For many experiments on suspensions of large p a r t i c l e s such as blood c e l l s , a small He-Ne l a s e r c o s t i n g only a few hundred d o l l a r s i s s u f f i c i e n t . The e s s e n t i a l o p t i c a l components can be kept t o as l i t t l e as one lens and two p i n h o l e s , though i n p r a c t i c e we use a more complicated real-image viewing system t o f a c i l i t a t e alignment of the s c a t t e r e d l i g h t and the l o c a l o s c i l l a t o r (5). In some cases the l o c a l o s c i l l a t o r may be taken from one or both of the spots formed by entry and e x i t of the l a s e r beam through the chamber. We g e n e r a l l y p r e f e r t o have a separate o p t i c a l path f o r the l o c a l o s c i l l a t o r which bypasses the s c a t t e r i n g chamber and then i s recombined with the s c a t t e r e d l i g h t , u s u a l l y a f t e r being r e f l e c ted from the chamber window. A v a r i a b l e attenuator i n the l o c a l o s c i l l a t o r path allows adjustment of the r a t i o o f the l o c a l o s c i l l a t o r i n t e n s i t y t o the detected s c a t t e r e d l i g h t i n t e n s i t y . T h i s r a t i o i s u s u a l l y i n the range from 10:1 t o 40:1. An e x t e r n a l l o c a l o s c i l l a t o r path i s e s s e n t i a l f o r s c a t t e r i n g angles above about 20°. We g e n e r a l l y work at angles between 50° and 60° when analyzing l a r g e p a r t i c l e s such as blood c e l l s , f o r which s p e c t r a l broadening due t o d i f f u s i o n i s n e g l i g i b l e . Much lower angles must be u t i l i z e d f o r the study of s o l u t i o n s of s m a l l e r p a r t i c l e s such as p r o t e i n s , i n order t o optimize the r a t i o o f the Doppler s h i f t t o the d i f f u s i o n - c o n t r o l l e d h a l f - w i d t h (2). However, f o r blood c e l l s t u d i e s we employ the higher s c a t t e r i n g angles t o i n c r e a s e the Doppler s h i f t f o r a given e l e c t r o p h o r e t i c v e l o c i t y . This adaptation i s e s s e n t i a l f o r performing e x p e r i ments at p h y s i o l o g i c a l i o n i c s t r e n g t h , where the e l e c t r o p h o r e t i c m o b i l i t i e s are lower and the a t t a i n a b l e f i e l d strength f o r a given t o l e r a b l e amount o f Joule h e a t i n g i s a l s o lower. The b e a t i n g d e t e c t i o n system r e q u i r e d f o r an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g measurement i s simpler and cheaper than the high-speed photon counting apparata necessary f o r many other l a s e r l i g h t s c a t t e r i n g a p p l i c a t i o n s . The photodetector can be an inexpensive p h o t o m u l t i p l i e r tube o r an even cheaper p h o t o r e s i s t o r or p h o t o v o l t a i c c e l l . The s i g n a l s are g e n e r a l l y i n the region between 1 Hz and 200 Hz, so a m p l i f i c a t i o n and spectrum a n a l y s i s are t r i v i a l low-speed problems. I t i s e s s e n t i a l t h a t the spectrum a n a l y s i s , whether done by a spectrum analyzer, an autoc o r r e l a t o r , or a d i g i t a l computer o r c a l c u l a t o r , be done i n r e a l time; i . e . , i n the minimum time r e q u i r e d f o r measurement with a given frequency r e s o l u t i o n . For example, to measure a spectrum with a r e s o l u t i o n of 1 Hz r e q u i r e s 1 sec. We need t h e r e f o r e apply the e l e c t r i c f i e l d f o r one second only, i f the data p r o c e s s i n g device can make use of a l l the information a v a i l a b l e during t h a t one second t o produce a complete spectrum. The a b i l i t y t o use pulsed f i e l d s i s an i n t r i n s i c advantage o f e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g over c l a s s i c a l e l e c t r o p h o r e s i s


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techniques, s i n c e i t permits the use o f a higher f i e l d s t r e n g t h , which i n many cases leads t o g r e a t e r e l e c t r o p h o r e t i c r e s o l u t i o n . We g e n e r a l l y apply constant-current pulses with a duty c y c l e o f about 1:10; the i n t e r v a l between pulses allows the d i s s i p a t i o n o f Joule heat. The pulses are of a l t e r n a t i n g p o l a r i t y so that there i s no net t r a n s p o r t o f mass during the experiment. Timing and t r i g g e r i n g are c o n t r o l l e d by a s p e c i a l l y designed clock c i r c u i t . The use o f a constant-current power supply i s important f o r maintaining a constant e l e c t r i c f i e l d during each p u l s e , s i n c e i t a u t o m a t i c a l l y c o r r e c t s f o r e l e c t r o d e p o l a r i z a t i o n e f f e c t s and changes i n v i s c o s i t y which accompany Joule h e a t i n g . The design o f the chamber i s a c r i t i c a l feature of the experiment. A l l chambers have two e l e c t r o d e s f o r a p p l i c a t i o n of the f i e l d and an o p t i c a l path f o r entry of the l a s e r beam and e x i t of the s c a t t e r e d l i g h t . However, a number of chamber c o n f i g u r a t i o n s have been employed, and each has i t s own advantages. I n t e r e s t e d persons are r e f e r r e d t o one of the reviews of t h i s technique (1,3,4,5) f o r d i s c u s s i o n and r e f e r e n c e s . We c u r r e n t l y have s e v e r a l d i f f e r e n t chambers i n use f o r various a p p l i c a t i o n s . Common features o f these chambers which we have found t o be important are low volume, e f f i c i e n t heat d i s s i p a t i o n , and subs t a n t i a l s e p a r a t i o n between the e l e c t r o d e and the s c a t t e r i n g r e g i o n , so t h a t p a r t i c l e s , bubbles, and/or l o c a l pH gradients formed a t the e l e c t r o d e s w i l l not be able t o reach the p o r t i o n of the s o l u t i o n which i s being viewed during the measurement. Although the experiment i s not easy, e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g i n i t s current s t a t e of development i s capable of measuring complete e l e c t r o p h o r e t i c m o b i l i t y d i s t r i b u t i o n s i n a few seconds. Once the apparatus i s s e t up, operation i s f u l l y automatic, and adaptation t o o n - l i n e p r o c e s s i n g of m u l t i p l e samples would be a s t r a i g h t f o r w a r d extension of current capabilities. In the absence of an e l e c t r i c f i e l d , the l i g h t s c a t t e r e d from macromolecules i n s o l u t i o n i s frequency-broadened by the random thermal motions of d i f f u s i o n . The measured spectrum i s a Lorentz i a n l i n e centered at the i n c i d e n t frequency, or, when b e a t i n g d e t e c t i o n i s used, centered at zero. A p p l i c a t i o n of the e l e c t r i c f i e l d causes the p a r t i c l e s to migrate, and, i f they a l l have the same e l e c t r o p h o r e t i c m o b i l i t y , the r e s u l t i n g spectrum i s a s h i f t e d L o r e n t z i a n l i n e whose width i s s t i l l determined by d i f f u s i o n and the magnitude of whose s h i f t i s d i r e c t l y r e l a t e d t o the e l e c t r o p h o r e t i c d r i f t v e l o c i t y , which, when d i v i d e d by the f i e l d s t r e n g t h , gives the e l e c t r o p h o r e t i c m o b i l i t y . This pred i c t i o n has been v e r i f i e d experimentally, and an example i s shown i n F i g u r e 2. The p o i n t s i n the spectrum shown i n Figure 2 are data from an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum on a d i l u t e s o l u t i o n of human carbon monoxyhemoglobin (1.6 mg/ml) at pH 9.5. The l i n e i s a L o r e n t z i a n f u n c t i o n f i t t o the s h i f t of the peak and with a h a l f - w i d t h corresponding t o the known d i f f u s i o n c o e f f i c i e n t of hemoglobin. We are c u r r e n t l y pursuing an


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i n t e r e s t i n the study of simultaneous d i f f u s i o n and e l e c t r o phoresis o f concentration f l u c t u a t i o n s under c o n d i t i o n s f o r which f l u c t u a t i o n s o f d i f f e r e n t species i n s o l u t i o n cannot be con s i d e r e d t o be uncoupled. This spectrum i s presented only t o show t h a t i n the simple case of uncoupled f l u c t u a t i o n s , the p r e d i c t i o n s o f the simple theory are observed. We have a l s o used ELS t o study the d i s s o c i a t i o n o f hemoglobin at high pH (8). I f there are more than one type o f macroions i n s o l u t i o n i n appreciable concentrations, and i f the d i f f e r e n t species have d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t i e s , then the e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum can be used t o detect and q u a n t i f y r e l a t i v e amounts of the s p e c i e s . A common example of a u s e f u l a n a l y t i c a l e l e c t r o p h o r e s i s determination i s the a n a l y s i s of human blood plasma. An e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum of human plasma i s shown i n Figure 3. Such a spectrum bears a strong resemblance t o plasma e l e c t r o p h o r e s i s by c l a s s i c a l t e c h niques, except t h a t the lower-mobility peaks, presumably due t o the various g l o b u l i n f r a c t i o n s , are enhanced with respect t o the l a r g e albumin peak because of t h e i r higher molecular weight. This p a r t i c u l a r measurement i s an extremely important c l i n i c a l t e s t , and some of our f r i e n d s i n i n d u s t r y t e l l us t h a t e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g may be cost-competitive with c l a s s i c a l methods f o r t h i s a p p l i c a t i o n . When b i o l o g i c a l p a r t i c l e s l a r g e r than p r o t e i n s , p a r t i c u l a r l y membranous p a r t i c l e s , are analyzed, i t i s observed t h a t there i s s u b s t a n t i a l e l e c t r o p h o r e t i c heterogeneity. Moreover, these l a r g e r p a r t i c l e s have correspondingly low d i f f u s i o n c o e f f i c i e n t s , and the d i f f u s i o n broadening i s t h e r e f o r e o f t e n i n s i g n i f i c a n t . For such p a r t i c l e s the e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum i s a determination of the e l e c t r o p h o r e t i c m o b i l i t y d i s t r i b u t i o n of the sample. As an i l l u s t r a t i o n I present the spectrum i n F i g u r e 4. The sample i n t h i s case was a mixture o f human and r a b b i t red blood c e l l s ; human c e l l s have the higher m o b i l i t y . The sharp r e s o l u t i o n i s a v i v i d demonstration t h a t ELS can be used t o detect s e v e r a l species simultaneously. The e l e c t r o p h o r e t i c m o b i l i t y o f red c e l l s i s q u i t e uniform, and i n a recent p u b l i c a t i o n with two other groups we have demonstrated t h a t pre vious r e p o r t s t h a t the e l e c t r o p h o r e t i c m o b i l i t i e s of red blood c e l l s decrease with age were erroneous (9). We have been using e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g t o study the surface p r o p e r t i e s of l i v i n g c e l l s and o r g a n e l l e s , and the remainder of t h i s l e c t u r e w i l l be a b r i e f summary o f some of our work i n t h i s area. One of the o r i g i n a l areas o f i n t e r e s t was the c h a r a c t e r i z a t i o n o f c e l l s i n v o l v e d i n the immune response, f o r which surface c h a r a c t e r i s t i c s are p a r t i c u l a r l y important. For example, lymphocytes are the white blood c e l l s i n v o l v e d with immunological r e c o g n i t i o n and response. Lymphocytes are u s u a l l y d i v i d e d i n t o two c a t e g o r i e s : Τ c e l l s , which are p r i m a r i l y i n volved with d i r e c t c e l l u l a r immunity, and Β c e l l s , which are r e s p o n s i b l e f o r the s y n t h e s i s of s p e c i f i c a n t i b o d i e s . We have


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Figure 2. Electrophoretic light-scattering spec trum (points) of carhoxyhemoglobin tetramers, 100 μΜ in heme. The experimental conditions are Ε = 88.8 V/cm, θ = 4.18°, bath temperature = 20.0°C, and a glycineNaOH-NaCl-EDTA buffer of ionic strength 0.01M and pH 9.5. The solid line is a theoretical curve for these conditions, assuming diffusion is the only source of spectral broadening with a diffusion coefficient D = 6.9 X 10' cm /sec and an electrophoretic mobility u = 2.74 X 10' cm /V-sec (7). 2 0

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Figure 3. Electrophoretic light-scattering spectrum of human blood plasma. Fresh human plasma was dialyzed and then diluted severalfold to final solution conditions of pH 9.1 and ionic strength 0.004. This spectrum was taken with a high field strength (183 V'/cm) to maximize the Doppler shift and at a low scattering angle (3.2°) to minimize the diffusion broadening of each peak. The Urge peak at the highest frequency can be identified as albumin from its relative magnitude and its electrophoretic mobility (3.9 X 10~ cm /V-sec). Positive identification of the peaks at lower mobility cannot be made from the Doppler spectrum alone, but the form of the spectrum is similar to the known electrophoretic pattern of normal human plasma (5). 4

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been studying human lymphocytes and t h e i r surface r e a c t i o n s , and we have been comparing them w i t h p a t h o l o g i c a l c o n d i t i o n s such as acute lymphocytic leukemia, i n which the lymphocytes, or more p r o p e r l y lymphoblasts, produced are malignant, d i v i d i n g c e l l s which have g r e a t l y reduced immunological c a p a c i t y . An e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum o f normal human lymphocytes i s shown i n Figure 5. The spectrum i s l a b e l l e d i n u n i t s of e l e c t r o p h o r e t i c m o b i l i t y , because f o r these c e l l s d i f f u s i o n broadening i s i n s i g n i f i c a n t , and there i s a one-to-one c o r respondence between frequency and m o b i l i t y . The s o l i d l i n e i s the spectrum from a normal sample o f lymphocytes i n one-tenthp h y s i o l o g i c a l - s a l t medium at pH 7.4. Note the bimodal c h a r a c t e r . To determine whether these two peaks could be a t t r i b u t e d t o Τ and Β c e l l s we prepared samples from which Τ or Β c e l l s had been s e l e c t i v e l y removed. The dotted l i n e i s the spectrum f o r t h i s sample from which Τ c e l l s had been s e l e c t i v e l y removed by c e l l r o s e t t i n g techniques. Note t h a t the h i g h e r - m o b i l i t y peak i s s e l e c t i v e l y diminished. By performing numerous experiments o f t h i s type we have e s t a b l i s h e d t h a t the h i g h e r - m o b i l i t y peak i s due p r i m a r i l y t o Τ c e l l s and the lower-mobility peak i s due p r i m a r i l y t o Β c e l l s (10) , which i s i n agreement with s e v e r a l groups who have obtained t h i s same r e s u l t and i n c o n f l i c t w i t h some groups who have claimed t h a t the two c e l l subtypes are e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e . Our method i s now by f a r the f a s t e s t means o f measuring the Τ c e l l / B c e l l r a t i o , which i s a parameter o f both research and c l i n i c a l i n t e r e s t . In a f u r t h e r attempt to c h a r a c t e r i z e these lymphocytes we have performed experiments t o determine the o r i g i n of the c e l l s u r f a c e charge and the d i s t i n c t i o n between c e l l subtypes on t h i s b a s i s . For example, s i a l i c a c i d (N-Acetylneuraminic acid) i s a ubiquitous source of charge i n c e l l membranes. S i a l i c a c i d can be removed by the a c t i o n o f the enzyme neuraminidase, and we have t r e a t e d lymphocytes with neuraminidase and analyzed separated subf r a c t i o n s . The r e s u l t s are i l l u s t r a t e d i n Figure 6. Again the s o l i d l i n e represents the whole f r a c t i o n o f the same sample seen i n the p r e v i o u s f i g u r e except a f t e r neuraminidase treatment. The m o b i l i t i e s are lower as expected. The dotted l i n e again r e p r e sents t h i s same sample from which Τ c e l l s had been s e l e c t i v e l y removed. Note t h a t the lower-mobility peak shows the only reduc t i o n i n i n t e n s i t y . By repeated experiments with both Τ and Β depleted samples we have demonstrated t h a t a f t e r neuraminidase treatment the Τ c e l l s , which were o r i g i n a l l y o f h i g h e r m o b i l i t y , become the lower-mobility f r a c t i o n , i n d i c a t i n g t h a t they have much more a v a i l a b l e s i a l i c a c i d on t h e i r s u r f a c e s . In f a c t the e l e c t r o p h o r e t i c d i s t i n c t i o n between the two types o f c e l l s i s even g r e a t e r a f t e r neuraminidase treatment. Comparison w i t h diseased s t a t e s i s i n t e r e s t i n g both p o s s i b l e development o f the e l e c t r o p h o r e t i c m o b i l i t y as i n d i c a t o r and f o r a c h a r a c t e r i z a t i o n o f the fundamental ences of the abnormal c e l l s . We have been p a r t i c u l a r l y

f o r the a clinical differ interested


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Figure 4. Electrophoretic light scat tering spectrum for a mixture of rabbit and human erythrocytes in approxi mately equal concentrations. Rabbit erythrocytes have the lower mobility. The measurement was made in an electro phoresis buffer which had an ionic strength of 0.0097. The electric field was 44 V/cm, the frequency range 200 Hz, and the scat tering angle 58°. The chamber tempera ture was 20°C. The mobility distributions for the two cell types are completely re solved (6).

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Journal of Immunology

Figure 5. A comparison of the electrophoretic mobility distributions (at 0.015M ionic strength) of a fresh human mononuclear, white-blood-cell sample before (solid line) and after (dotted line) E rosette depletion (T-cell depletion). The whole sample (solid line) contained 44% cells which form E rosettes and 32% cells which form Ε AC rosettes (primarily Β cells). The horizontal axis indicates the magnitude of the electrophoretic mobility since the Doppler tech nique does not determine the sign of the mobility, which for these cells is nega tive. The vertical axis is approximately proportional to cell number (10). AET

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ι

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Figure 6. A comparison of the electrophoretic mobility distributions (at 0.015M ionic strength) for the same pair of samples shown in Figure 5 after both had been treated with neuraminidase. Again, the solid line represents the whole sample and the dotted line represents the E -rosette-depleted (T-cell depleted) sample. Τ cells are therefore represented primarily in the low-mobility peak between 0.5 and 1.0 mobility units (10). AET


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i n comparisons with leukemic c e l l s (10,11). Electrophoretic comparison of normal lymphocytes and c e l l s obtained i n the same way from p a t i e n t s w i t h acute lymphocytic leukemia i s shown i n Figure 7. The s o l i d l i n e i s a normal sample w i t h i t s charac t e r i s t i c bimodal appearance. The dotted l i n e i s a sample of leukemic c e l l s . Note t h a t t h e i r mode m o b i l i t y i s lower and t h a t the d i s t r i b u t i o n i s narrower, w i t h no bimodal c h a r a c t e r , i n d i c a t i v e o f the l a c k o f d i f f e r e n t i a t i o n of these c e l l s . The m o b i l i t y of leukemic c e l l s i s q u i t e v a r i a b l e , ranging from 5% t o 25% lower than normal c e l l s at t h i s s a l t c o n c e n t r a t i o n (0.015 M). Whether the m o b i l i t y i s a meaningful c l i n i c a l i n d i c a t o r has not y e t been determined, though a group i n France u s i n g c l a s s i c a l micro e l e c t r o p h o r e s i s has r e c e n t l y r e p o r t e d t h a t i t may be (12). We have a l s o s t u d i e d the response of leukemic c e l l s t o neuraminidase. Those experiments are summarized i n Figure 8. The s o l i d l i n e i s the normal sample and the d o t t e d l i n e i s the leukemic sample. Note t h a t the leukemic sample has an extremely narrow m o b i l i t y d i s t r i b u t i o n and t h a t i t f a l l s s l i g h t l y below the Τ c e l l mobility. This s l i g h t difference i s reproducible. Recall t h a t the leukemic c e l l s had a m o b i l i t y s i m i l a r t o the Β c e l l m o b i l i t y b e f o r e treatment; a f t e r neuraminidase treatment they show a m o b i l i t y f a r from Β c e l l s and c l o s e r t o Τ c e l l s . Clearly the surface o f the leukemic c e l l i s markedly d i f f e r e n t from either Τ or Β c e l l s . T h i s f a c t has a l s o been i l l u s t r a t e d by experiments we have performed on the i o n i c strength dependence o f the m o b i l i t i e s , which i s d i f f e r e n t f o r leukemic c e l l s than f o r Τ or Β c e l l s . In f a c t , leukemic c e l l s have a mode m o b i l i t y which i s about the same as normal samples at p h y s i o l o g i c a l i o n i c strength. These experiments on lymphocytes and leukemic lymphoblasts represent an almost s t r i c t l y a n a l y t i c a l approach t o the a p p l i c a t i o n o f the technique. Are there more fundamental questions which we can address? I wouldn't ask the question i f the answer were not yes, and i n p a r t i c u l a r I want t o describe some e x p e r i ments we have been performing on the general q u e s t i o n of the r o l e of e l e c t r o s t a t i c f o r c e s i n c e l l - c e l l i n t e r a c t i o n s . A l l b i o l o g i c a l membranes are negative and i t i s reasonable t o expect e l e c t r o s t a t i c r e p u l s i o n between them. However, we know t h a t many d i f f e r e n t kinds o f c e l l adhesion and aggregation r e a c t i o n s occur f r e q u e n t l y . This i s much e a s i e r t o accept when we a p p r e c i a t e t h a t the charges on c e l l s are screened e x p o n e n t i a l l y by the counterions i n s o l u t i o n , w i t h a space constant equal t o the r e c i p r o c a l o f the Debye-Huckel constant, which a t p h y s i o l o g i c a l i o n i c strength i s about 8 A. S t i l l one sees and hears many arguments i n c e l l adhesion problems on the r o l e o f e l e c t r o s t a t i c f o r c e s , so we have s e t about the task o f measuring c e l l charge and c o r r e l a t i n g with aggregation phenomena. The f i r s t system we s t u d i e d was the g r a n u l o c y t e s , o r p o l y morphonuclear white b l o o d c e l l s , which are the phagocytes respon s i b l e f o r e a t i n g and d i g e s t i n g f o r e i g n m a t e r i a l . These c e l l s


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I

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Figure 7. A comparison of the electrophoretic mobility distributions for normal (solid line) and leukemic (dotted line) human mononuclear white blood cells at 0.015M ionic strength. The leukemic cells have a distinctly lower mode mobility than the normal cells. In this case, the leukemic cell distribution almost coincides with that portion of the normal distribution that has been identified as Β cells (10).


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Figure 8. A comparison of the electrophoretic mobility distributions of the same pair of samples shown in Figure 7, after neuraminidase treatment. Again, the solid line represents the normal sample and the dotted line represents the leukemic sample. The mobility of the leukemic cells is reduced by a much larger frac tion than that of the Β cells, so that the leukemic-cell mobility is now slightly lower than the Τ-cell mobility. Thus the leukemic cells more nearly resemble Τ cells in their response to neuraminidase treatment (10).


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concentrate i n areas o f i n f e c t i o n i n response t o s o l u b l e p r o t e i n f a c t o r s c a l l e d lymphokines, which are s e c r e t e d by lymphocytes i n a f f e c t e d areas. One p o s s i b i l i t y advanced was t h a t the lymphok i n e s , c a l l e d LIF f o r leukocyte i n h i b i t i o n f a c t o r , b i n d t o the surface o f the g r a n u l o c y t e s , reduce t h e i r charge, and thereby cause them t o adhere t o each other and t o other s u r f a c e s i n the area. In experiments which are not y e t p u b l i s h e d , we have compared the e l e c t r o p h o r e t i c m o b i l i t i e s of granulocytes b e f o r e and a f t e r treatment with LIF and before and a f t e r treatment with cont r o l f r a c t i o n s i s o l a t e d i n the same way as the L I F . N e i t h e r the LIF nor the c o n t r o l s induced any measurable change i n the e l e c t r o p h o r e t i c m o b i l i t i e s o f the granulocytes at p h y s i o l o g i c a l i o n i c s t r e n g t h (6). T h e r e f o r e , the charge-reduction mechanism d e r i v e s no support from our data. But what i f the s u r f a c e charge were reduced? Would t h i s nece s s a r i l y cause an i n c r e a s e i n aggregation? To address t h i s quest i o n , we have i n i t i a t e d a study on mouse f i b r o b l a s t s ( s t r a i n 3T3 MIT), which are d i v i d i n g connective t i s s u e c e l l s . These experiments were a l s o done at p h y s i o l o g i c a l i o n i c s t r e n g t h i n phosphate b u f f e r e d s a l i n e t o approximate as c l o s e l y as p o s s i b l e the r e l e v a n t p h y s i o l o g i c a l parameters. We considered three d i f f e r e n t types o f m o d i f i c a t i o n s o f the c e l l s u r f a c e s : neuraminidase treatment, v i r a l t r a n s f o r m a t i o n , and urea treatment. Electrop h o r e t i c m o b i l i t y histograms were measured by e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g , and the degree of change o f s u r f a c e charge was c o r r e l a t e d with the c e l l aggregation r a t e f o r the same c e l l s measured i n a hemacytometer by our c o l l a b o r a t o r s , Morris Karnovsky and Tom Wright o f the Harvard Medical School. The r e s u l t s have been d e s c r i b e d i n the t h e s i s of Barton Smith (6) and w i l l be p u b l i s h e d i n the near f u t u r e . To summarize, we found t h a t neuraminidase treatment o f these c e l l s lowers the s u r f a c e charge and i n c r e a s e s the aggregation rate by more than a f a c t o r o f two. V i r a l t r a n s f o r m a t i o n with SV-40 and polyoma v i r u s e s produces o n l y a few percent decrease i n the average s u r f a c e charge and y e t i n c r e a s e s the aggregation rate by more than a f a c t o r of t h r e e . Treatment o f the transformed c e l l s w i t h neuraminidase produced a l a r g e r e d u c t i o n i n t h e i r average e l e c t r o p h o r e t i c m o b i l i t y but no p e r c e p t i b l e change i n t h e i r r a t e o f aggregation. F i n a l l y t r e a t ment with 0.20 M urea produced no change i n the e l e c t r o p h o r e t i c histogram but i n c r e a s e d the rate o f aggregation by more than a f a c t o r o f t h r e e . We can only conclude that f o r t h i s system the c e l l s u r f a c e charge does not c o r r e l a t e w e l l with the p r o p e n s i t y o f the c e l l s t o adhere t o each other. C e l l adhesion must be understood on the b a s i s o f more d e t a i l e d molecular r e c o g n i t i o n and with an accounting f o r the balance between the a t t r a c t i v e van der Waals f o r c e s and the screened coulombic r e p u l s i o n s . The f i n a l type of study which I would l i k e t o d e s c r i b e i s an i n v e s t i g a t i o n i n t o the process o f v e s i c u l a r s e c r e t i o n . The sec r e t i o n of v a r i o u s hormones and neurotransmitters i n v o l v e s the r e l e a s e o f these molecules from s p h e r i c a l v e s i c l e s i n which they


WARE


Figure 9. Electrophoretic mobilities of chromaffin granules vs. con centration of Ca , Mg" , and Mg-ATP; and electrophoretic mobility of plasma membrane vesicles vs. concentration of Ca . +2

2

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Mobilities are negative, viscosity corrected to pure water at 20°C, and are given in units of μm-cm/V-sec. The ionic strength of the suspension medium was 15 mM and the pH was 6.9. Note the Ca and Mg have identical effects on the mobilities, and hence on the surface charge, of chromaffin granules. +2


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are s t o r e d . The s e c r e t o r y v e s i c l e s are thought t o r e l e a s e t h e i r contents by f u s i n g with the plasma membrane o f the c e l l i n which they r e s i d e . T h i s f u s i o n i s g e n e r a l l y t r i g g e r e d by an i n f l u x o f calcium i o n , which i s s p e c i f i c f o r t h i s process. We have been i n v e s t i g a t i n g the e f f e c t s o f calcium and magnesium ions on the surface charge and aggregation o f s e c r e t o r y v e s i c l e s . As an example I show the r e s u l t o f one such study on the chromaffin granules from the adrenal medulla, i n t h i s case from a cow. These granules are v e s i c l e s which c o n t a i n a d r e n a l i n and noradrenalin. They are e a s i l y i s o l a t e d and p u r i f i e d . We have t i t r a t e d them with C a and M g and have monitored the e f f e c t o f these ions on granule s u r f a c e charge by e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g . The r e s u l t s o f t h i s t i t r a t i o n are shown i n F i g u r e 9. Broken l i n e s represent the t i t r a t i o n o f granules and the s o l i d l i n e shows a t i t r a t i o n o f v e s i c l e s formed from the plasma mem branes o f the chromaffin c e l l . One o f the most i n t e r e s t i n g r e s u l t s o f t h i s study was the f i n d i n g t h a t C a and M g have the same a f f i n i t y f o r the granule s u r f a c e , which means t h a t the s p e c i f i c r o l e o f calcium i n inducing e x o c y t o s i s cannot be d e t e r mined by i t s b i n d i n g constant. We have a l s o performed e x p e r i ments i n which mixtures o f the granules and the plasma membrane v e s i c l e s are observed f i r s t s e p a r a t e l y and then i n the same s o l u t i o n t o d e t e c t aggregation o f the two s p e c i e s . Our e x p e r i ments show no aggregation below 1 mM C a and considerable aggregation above t h a t c o n c e n t r a t i o n . We are c u r r e n t l y pursuing experiments o f t h i s type t o study the neurosecretory process. I t r u s t t h a t these examples serve adequately t o i l l u s t r a t e the various types o f a p p l i c a t i o n s f o r which e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g can be u s e f u l . Probably the two most u s e f u l techniques i n b i o p h y s i c a l s t u d i e s have been spectroscopy and e l e c t r o p h o r e s i s . This new technique, which i s a spectroscop i c a l l y - d e t e c t e d e l e c t r o p h o r e s i s , combines the advantages o f speed, accuracy, r e s o l u t i o n , and o b j e c t i v i t y and w i l l , I b e l i e v e , be a p p l i e d t o an i n c r e a s i n g number o f important problems i n b i o l o g y and s u r f a c e chemistry i n the years t o come. + 2

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LITERATURE CITED 1.

Ware, B. R., " A p p l i c a t i o n s o f Laser Velocimetry in Biology and Medicine," in Chemical and Biochemical Applications of Lasers, C. B. Moore, ed., Chapter 5, Academic Press, New York, 1977.

2.

Ware, B. R., and F l y g a r e , W. Η., "The Simultaneous Measure ment o f the E l e c t r o p h o r e t i c M o b i l i t y and D i f f u s i o n C o e f f i c i e n t in Bovine Serum Albumin S o l u t i o n s by L i g h t S c a t t e r i n g , " Chem. Phys. Lett. (1971) 12, 81.

3.

Ware, B. R., " E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Adv. Interface Sci. (1974) 4, 1.


Colloid

7.

WARE


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4.

F l y g a r e , W. Η., Ware, B. R., and H a r t f o r d , S. L., " E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " in Molecular Electro-Optics, C. T. O'Konski, ed., Chapter 9, Marcel Dekker, Inc., New York, 1976.

5.

Smith, Β. Α., and Ware, B. R., "Apparatus and Methods for Laser Doppler E l e c t r o p h o r e s i s , " in Contemporary Topics in Analytical and Clinical Chemistry, Hercules et al., eds., Plenum Press, New York, in press.

6.

Smith, Β. Α., "The Study o f Cell Surface Charge by E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Ph.D. Thesis (1977) Harvard U n i v e r s i t y , Cambridge, Massachusetts.

7.

Haas, D. D., and Ware, B. R., "Design and Construction o f a New E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g Chamber and A p p l i c a t i o n s t o S o l u t i o n s o f Hemoglobin," Anal. Biochem. (1976) 74, 175.

8.

Haas, D. D., and Ware, B. R., " E l e c t r o p h o r e t i c M o b i l i t i e s and D i f f u s i o n C o e f f i c i e n t s o f Hemoglobin a t High pH," submitted for publication.

9.

Luner, S. J . , Szklarek, D., Knox, R. J., Seaman, G. V. F., Josefowicz, J . Υ., and Ware, B. R., "Red Cell Charge is Not a Function o f Cell Age," Nature (London), (1977) 269, 719.

10. Smith, Β. Α., Ware, B. R., and Yankee, R. Α., " E l e c t r o p h o r e t i c M o b i l i t y D i s t r i b u t i o n s o f Normal Human Τ and Β Lymphocytes and o f P e r i p h e r a l Blood Lymphoblasts in Acute Lymphocytic Leukemia: E f f e c t s o f Neuraminidase and o f Solvent I o n i c Strength," J . Immunol. (1978) 120(3), 921. 11. Smith, Β. Α., Ware, B. R., and Weiner, R. S., " E l e c t r o p h o r e t i c D i s t r i b u t i o n s o f Human P e r i p h e r a l Blood Mononuclear White C e l l s from Normal Subjects and from P a t i e n t s with Acute Lymphocytic Leukemia," Proc. Natl. Acad. Sci. (USA) (1976) 73, 2388. 12. Sabolovic, D., Pompidou, Α., and Amiel, J . L., "Blood Lympho cytes in Acute Lymphoid Leukaemia in Remission and in Relapse. P r e d i c t i v e Value o f E l e c t r o p h o r e t i c M o b i l i t y and Refringence," Biomed. (1975) 23, 283. 13. S i e g e l , D. P., Ware, B. R., Green, D. J . , and Westhead, E. W., "The E f f e c t s o f Ca and Mg on the Surface Charge o f Chro maffin Granules Measured by E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Biophys. J. (1978) 22, 341. +2

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RECEIVED August 7, 1978.


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