Macromolecular Mapping Research Articles

Global distribution and surface activity of macromolecules in offline simulations of marine organic chemistry

Organic macromolecules constitute a high percentage of remote sea spray. They enter the atmosphere through adsorption onto bubbles followed by bursting at the ocean surface, and go on to influence the chemistry of the fine mode aerosol. We present a global estimate of mixed-layer macromolecular distributions, driven by offline marine systems model output. The approach permits estimation of oceanic concentrations and bubble film surface coverages for several classes of organic compound. Mixed layer levels are computed from the output of a global ocean ecodynamics model by relating the macromolecules to standard biogeochemical tracers. Steady state is assumed for labile forms, and for longer-lived components we rely on ratios to existing transported variables. Adsorption is then represented through conventional Langmuir isotherms, with equilibria deduced from laboratory analogs. Open water concentrations locally exceed one micromolar carbon for the total of proteins, polysaccharides and refractory heteropolycondensates. The shorter-lived lipids remain confined to regions of strong biological activity. Results are evaluated against available measurements for all compound types, and agreement is generally well within an order of magnitude. Global distributions are further estimated for both fractional coverage of bubble films at the air–water interface and the two-dimensional concentration excess. Overall, we show that macromolecular mapping provides a novel tool for the comprehension of oceanic surfactant patterns. These results may prove useful in planning field experiments and assessing the potential response of surface chemical behaviors to global change.

Two-dimensional polyacrylamide gel electrophoresis of rabbit skeletal muscle microsomal proteins

Since the application of polyacrylamide gel electrophoresis to the study of membrane proteins, there have been a number of reports on the protein composition of skeletal muscle microsomes prepared by differential pelleting [l-7] and by density gradient centrifugation [g-10] . These electrophoretic separations of the solubilised membrane proteins have been in one-dimension. There is general agreement that the principal protein component of these preparations is a protein of mol. wt 100 000. Furthermore this component has been shown to be the Ca++ transport ATPase of the sarcoplasmic reticulum [ 1,2,8,9] . Estimates of the amount of this protein present have varied from 3570%. In addition to this major protein five other proteins and a proteolipid have been separated [4,11]. These other proteins have been postulated to play roles associated with the calcium transport function of the sarcoplasmic reticulum [3,4,11,12]. This report describes the use of two-dimensional polyacrylamide gel electrophoresis in the study of membrane proteins from four microsomal fractions prepared by zonal centrifugation [ 10,131. The twodimensional separation consists of electrophoresis in the first dimension in 5.2% gels followed by electrophoresis in the second dimension in gels having a 627% gel gradient. This method is especially useful in the macromolecular mapping of multicomponent mixtures [14,15] and is a novel approach in the study of membrane proteins. The present work is part of a continuing study of rabbit skeletal muscle microsomes. The results reported here show that muscle microsomes contain many more proteins than was previously believed.

Colinearity of protein patterns in polyacrylamide gel electrophoresis

1. 1. Quantitative evaluation of polyacrylamide gel electrophoretic results is essential for comparative macromolecular mapping. 2. 2. Comparison of electrophorograms arising in a continuous signal ∗ ∗ Description of the term ‘signal’ Signal represents the message on changing protein patterns in polyacrylamide gels intended for comparison. Continuous or analogous signal describes our insight into changing protein pattern arising in a continuously changing system e.g. development. This signal is presented as a family of basic signals obtained by densitometric recordings of each protein pattern, and represents continuous monitoring of the single parameter i.e. protein composition of the complex system. Discontinuous signal, to the contrary, describes changes between the compared protein patterns, obtained with samples taken from the same horizontal level and which are therefore devoid of successive relationship among them e.g. population genetics. is possible when sufficient colinearity of the resolved bands is achieved. 3. 3. Comparison of protein patterns arising in a discontinuous signal must rely upon statistical evaluation of densitometric recordings. 4. 4. Examples of the two different types of comparison are given from the study of rat ontogenesis.

Polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0 degrees and 25 degrees C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.

Pore gradient electrophoresis

Gel gradient electrophoresis has been proposed as a method for improving resolution and for measuring molecular size. Margolis and Slater have suggested that each macro-ion will reach a “pore limit” or “dead stop” and that the protein pattern would remain essentially stable thereafter. However, no quantitative treatment of the migration of charged macromolecules in a gel gradient has been reported to date. The present report provides an analysis of the behavior of macro-ions in both linear and nonlinear gel gradients. This is made possible by combining Ferguson's relationship for electrophoretic mobility versus gel concentration with the equation describing the gel gradient. Solution of these two equations yields the instantaneous velocity and position for each band as a function of time. This analysis provides insight into situations in which pore gradients may improve resolution and conditions for optimal resolution of multicomponent systems (macromolecular mapping). Position and instantaneous velocity of migration for any macromolecule on gel concentration (“pore”) gradient electrophoresis may be calculated for a linear gradient: χ= log e(ba 2u ot + exp (bT o)) − bT o ba 2 (iva) ν= u o (ba 2u ot + exp (bT o)) (va) This makes it possible to calibrate or standardize pore gradient electrophoresis. This analysis indicates that the proteins do not come to a “pore limit” or “dead stop.” Pore gradient electrophoresis is not indicated for analysis or separation of one, two, or three components, nor for charge fractionation. It is useful for simultaneous analysis of components of multicomponent mixtures (macromolecular mapping); and a “transverse” gradient presents a promising approach to obtaining Ferguson plots.