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Protein Sci. Jun 2002; 11(6): 1558–1564.
PMCID: PMC2373635

Covalent cross-linking of proteins without chemical reagents


A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.

Keywords: Protein cross-linking, lyophilized protein, zero-length cross-linking, covalent cross-linking, in vacuo modification

Intermolecular covalent cross-linking of functional groups in proteins has proved to be a very useful approach in the study of structure–function relationships in proteins, especially in multiprotein complexes (Fancy 2000; Phizicky and Fields 1995; Lundblad 1994). This technique also has many practical applications, particularly in improving the stability of the quaternary structure of proteins. The subunits of hemoglobin, for example, have been cross-linked to reinforce active structure and produce a more stable oxygen transporter (Manning et al. 1991; Chang 1998; White and Olsen 1987; Jones et al. 1996). Reaction with homobifunctional and heterobifunctional reagents with variable spacing between the reactive groups has been the most widely used strategy to achieve protein cross-links (Lundblad 1994). In particular, glutaraldehyde and bis-imido esters have been widely used because of their ability to form stable derivatives with the amino groups that are present in relatively high abundance in most proteins.

Zero-length cross-linking, in which peptide chains are covalently linked through existing functional groups without the incorporation of a spacer group, has been extensively used to immobilize proteins on non-protein matrices. This is usually accomplished by activation of carboxyl groups on a protein with a water soluble carbodiimide followed by coupling with an amino group on the support to form a stable amide bond (Lundblad 1994). Such cross-links between interacting proteins have been obtained by reaction with carbodiimide in the multi-protein electron transport system (Mauk and Mauk 1989), but there are very few other reported cases in which such zero-length cross-links have been achieved. Zero-length cross-linking of this type is most likely to occur when carboxyl groups are situated in proximity to amino groups, perhaps in salt linkages. Zero-length cross-linking can therefore provide information about local protein–protein interactions and has the advantage that no foreign group is introduced into the protein.

Ionic interactions between an amino and a carboxyl group often occur in protein–protein interactions (Milligan 1996). The present investigation was undertaken to determine whether advantage could be taken of this interaction to promote the formation of zero-length amide cross-links. The rationale for undertaking this study was derived from the observation that protonated amino groups in lyophilized proteins reacted to a significant extent with iodomethane in vacuo to form trimethylated amino derivatives (Vakos et al. 2001). The most likely explanation for such an apparently unfavorable reaction is that it is driven by the removal of one of the products, namely, hydrogen iodide, under vacuum. The condensation of ammonium and carboxylate groups to form an amide bond is thermodynamically unfavorable. However, if the reaction takes place in vacuo and water is removed, the subsequent formation of an amide bond should become more favorable. In addition, heating the lyophilized protein under vacuum should facilitate the removal of water and increase the rate of the condensation reaction, thereby promoting intermolecular cross-linking. In the present communication, we describe the results of such a study in which proteins, lyophilized under various conditions, are simply placed in a glass container under vacuum and heated in an oven.


Figure 1 [triangle] shows the extent of cross-linking achieved when RNase A, lyophilized at pH 7, is cycled through successive 24-h heating periods at 85°C under vacuum, redissolved, then relyophilized for a total of four heating periods. A strong band with an apparent molecular mass of approximately 28 kD, expected for the dimer, becomes evident after only one heating period of 24 h in vacuo and intensifies as the heating time increases to 96 hr. Weaker bands that correspond to the expected masses of RNase A trimers (~42 kD) and tetramers (~56 kD) are also clearly visible. Unfortunately, in this particular gel the lane with the molecular weight markers has run anomalously as a result of an edge effect and the expected masses due from the dimer and trimers do not correspond exactly to the masses indicated by these markers. Samples that had cycled through relyophilization before each heating trial show no increase in dimer formation compared with those kept continuously heating under vacuum for an equal length of time (Fig. 1 [triangle], lane 7). Maximum dimer formation was obtained after 96 h of heating in vacuo. The SDS-PAGE protein loading was high (20 μg), and even in the untreated sample (Fig. 1 [triangle], lane 2) a small amount of dimeric RNase is visible, probably formed during the initial lyophilization of the sample. A band with a slightly higher apparent molecular mass than monomeric RNase A is also evident and is attributable to an RNase B impurity present in the preparation (as per product description, Sigma-Aldrich, product number R4875).

Fig. 1.
SDS-PAGE of successive cycles of solubilization of 10 mg of RNase A at pH 7.0, lyophilization, then heating in vacuo for 24 hr. Total protein load per lane is 20 μg. Lane 1: low range molecular weight marker; lane 2: lyophilized RNase A, no heating ...

Very similar results were obtained with lysozyme (Fig. 2 [triangle]) where, again, the dimer is the major product with a smaller amount of higher oligomer. As in the case of RNase A, a small amount of dimeric lysozyme is visible in the untreated sample (Fig. 2 [triangle], lane 2). Lyophilized bovine serum albumin, hemoglobin, and chymotrypsin treated under the same conditions showed approximately the same extent of dimer formation (data not shown).

Fig. 2.
SDS-PAGE of successive cycles of solubilization of 10 mg of lysozyme at pH 7.0, lyophilization, then heating in vacuo for 24 hr. Total protein load per lane is 20 μg. Lane 1: low range molecular weight marker; lane 2: lyophilized lysozyme, no ...

The amount of RNase A dimer (Fig. 1 [triangle]) was estimated by the pixel-counting application ImageQuaNT 5.1 and size exclusion chromatography (SEC) (Fig. 3 [triangle]). RNase A dimer yields varied from 20% to 30% of the total treated protein depending on the length of the heating period. The elution profile of the RNase A heated in vacuo for 96 h (Fig. 3B [triangle]) has peaks with molecular masses of 28,000 Da, and 14,000 Da, corresponding to the RNase A dimer and monomer, respectively. From the total area under the peaks, the amount of dimer present was found to be approximately 30% of the total protein, in agreement with the estimate by pixel counting of the gel photographs. This isolated dimer was shown by an RNase in-gel activity assay to retain the activity of the monomer.

Fig. 3.
Size-exclusion fast performance liquid chromatography (FPLC) of in vacuo cross-linked RNase A. Separation of RNase A cross-linked products achieved with two Superdex G75 HR 10/30 columns in tandem using a mobile phase of 0.2 M Na2HPO4 and 0.15 M NaCl ...

The effect of pH on the extent of cross-linking in vacuo was determined by preparing RNase A solutions with pH values varying from 3.0 to 10.0, then lyophilizing and heating under vacuum. It was found that neutral to slightly alkaline pH values, that is, pH 7.0–9.0, favor the formation of dimer (Fig. 4 [triangle]).

Fig. 4.
The effect of pH on the extent of RNase A dimerization. RNase A samples of 10 mg/mL were adjusted to pH values 3.0 to 10.0 with 1.0 N NaOH, lyophilized, then cross-linked under vacuum. A 10-μg sample of the treated protein was subjected to SDS-PAGE. ...

The effect of cations present as counter-ions on the extent of cross-linking in vacuo was determined by addition of excess LiCl or CsCl followed by dialysis. The pH of the samples before lyophilization was adjusted with LiOH or CsOH. It was found that these counter-ions had no effect whatsoever on the extent of dimerization (data not shown).

The effect of lyophilization of RNase A in the presence of an excipient, trehalose, is shown in Figure 5 [triangle]. As the amount of trehalose present in the lyophilized sample increases, the amount of RNase A dimer produced decreases. At a 1 : 1 w/w ratio, trehalose appears to prevent any dimer formation, because only a trace of dimer similar to that observed in untreated samples is present.

Fig. 5.
Cross-linking of RNase A in the presence of trehalose at pH 7.0. RNase A (10 mg) was co-lyophilized with different amounts of trehalose and heated under vacuum for 96 h. A 20-μg sample of the treated protein was subjected to SDS-PAGE. Lane 1: ...

The results obtained when equal amounts (w/w) of RNase A and lysozyme were co-lyophilized are shown in Figure 6 [triangle]. Three bands are visible corresponding to the cross-linked dimeric RNase, cross-linked dimeric lysozyme, and the heterogeneously cross-linked lysozyme/RNase product. A higher extent of heterogeneous cross-linking than that shown could be obtained using longer heating times; however, the three cross-linked products could not be visualized as distinct bands on SDS-PAGE because of the high intensity of the resulting bands. RNase A was also co-lyophilized with high molecular weight polylysine or polyglutamic acid (1 : 1 w/w ) at pH 7.0. It was found that the high molecular weight fractions collected from SEC contained no free monomeric or dimeric RNase and had a high RNase activity in an RNase in-gel assay (data not shown).

Fig. 6.
Heterogeneous cross-linking of RNase A and lysozyme. RNase A (10 mg) was co-lyophilized with lysozyme (10 mg) at pH 7.0, heated under vacuum for 48 hr, and 15 μg of the treated protein was subjected to SDS-PAGE. Lane 1: RNase A (pH 7.0) alone ...

Two chemically modified RNase A samples were prepared, one in which the amino groups were dimethylated by reductive methylation and the other in which the carboxyl groups were modified by amidation with carbodiimide and glycinamide. The modified proteins were lyophilized and heated in vacuo under the same conditions as the unmodified protein. In both cases, no dimerization was observed (Fig. 7 [triangle]).

Fig. 7.
SDS-PAGE showing the effect of chemical modification of RNase A (15 mg/mL) on in vacuo cross-linking. Total protein loaded per lane is 10 μg. (A) Lane 1: RNase A lyophilized at pH 9.0 with no in vacuo treatment; lane 2: RNase A lyophilized at ...

The RNase A dimer was isolated by SEC and subjected to electrospray mass spectrometry (Fig. 8 [triangle]). The major peak in the spectrum occurs at 27,345 mass units corresponding to twice the mass of the monomer (13,682 ± 1 mass units) minus 18 mass units, that is, the loss of one water molecule, which shows that only one amide cross-link is present in the dimer. There is also a trace amount of a dimer peak at 27,327 mass units resulting from the loss of two water molecules and the formation of two amide cross-links. There is another significant peak at 27,361 mass units and a minor peak at 27,377 mass units, 16 and 32 mass units above the major dimer peak, respectively. The mass spectrum of RNase monomer used to prepare the dimer also shows a component 16 mass units greater than the monomer, which is probably a result of the presence of some RNase with methionine sulfoxide in the preparation. These two peaks, therefore, are probably a result of covalent dimers formed by condensations of molecules of RNase containing a residue of methionine sulfoxide.

Fig. 8.
Deconvoluted electrospray mass spectrum of the RNase A dimer produced by in vacuo cross-linking.


Our hypothesis is that the covalent cross-linking observed by the in vacuo procedure described occurs by formation of amide linkages that result from the condensation of a protonated amino group with a negatively charged carboxyl, and this condensation reaction is driven by the removal of water. When either the amino groups or the carboxyl groups are modified, by reductive methylation or amidation reactions, respectively, in vacuo cross-linking of the lyophilized protein does not occur. This result is consistent with the involvement of amino and carboxyl functions in the cross-linking. The observation that cross-linking is optimal when proteins were lyophilized at neutral to slightly alkaline pH values, that is, pH 7 to 9.0, in which amino and carboxyl groups are expected to be in their positively and negatively charged states, respectively, is also consistent with our hypothesis. The finding that RNase A could be covalently cross-linked to polylysine and polyglutamic acid polymers by the in vacuo procedure provides further evidence that the cross-linking is occurring by the formation of amide bonds between interacting ammonium and carboxylate groups.

Lyophilization of proteins removes most of the solvation shell separating individual molecules in solution, which results in direct interaction of the protein molecules. Excipients such as trehalose have often been added to protein solutions before lyophilization to replace this solvent shell and thereby stabilize the protein (Zaks 1992). In the case of RNase A, it was found that as the trehalose/RNase A ratio in the lyophilization mixture increases, the amount of dimer formation decreases. This result indicates that direct interaction of the protein molecules is required for in vacuo cross-linking and is also consistent with the formation of amide bonds between interacting ammonium and carboxylate groups.

The evidence obtained from the conditions required for successful in vacuo cross-linking and from the effects of chemical modification provide strong support for an amide cross-link. Nevertheless, this evidence is not definitive concerning the nature of the chemical cross-link. Mass spectroscopic analysis of the RNase A dimer from the in vacuo procedure shows that the covalent dimerization results from a condensation reaction with the concomitant loss of a water molecule. Given the evidence for the involvement of carboxyl groups, there are only three possibilities as to how such cross-linking can occur with the loss of a water molecule: (1) condensation of two protonated carboxyl groups to form an anhydride; (2) condensation of a protonated carboxyl with a hydroxyl group of a side chain to form an ester; or (3) condensation of a deprotonated carboxyl group with a protonated amino group to form an amide. For proteins lyophilized at neutral or slightly alkaline pH values, in which optimal in vacuo cross-linking occurs, the carboxyl groups are expected to be deprotonated and could not take part in anhydride or ester formation with the loss of a water molecule. Furthermore, covalent dimers formed by anhydrides or ester linkages would not survive the conditions for preparing and running SDS gels, namely, high temperatures in the presence of mercaptoethanol at an alkaline pH value. Therefore, the result of the mass spectroscopic analysis of the RNase A dimer combined with all the other evidence leaves no conclusion other than that the in vacuo dimerization occurs via amide bond formation. From the mass of the dimer, it can also be concluded that there are no other modifications that occur to the protein.

The yield of dimer obtained with RNase was approximately 30%. A possible explanation for this apparent limit is the presence of counter-ions, especially Na+, in association with carboxyl groups. This association could obstruct the salt-bridge interactions between protein molecules and reduce cross-linking efficiency. However, the presence of small or large cations, such as Li+ or Cs+, had no effect on the dimer yield. Thus, although the reason that dimer production in these experiments was limited to approximately 30% of the total protein remains obscure, the presence of cations associated with the lyophilized protein does not appear to be a factor.

It is expected that in vacuo cross-linked proteins will retain their biological activity when returned to an aqueous environment because this is the case for most lyophilized proteins. The RNase A dimer is active and RNase A cross-linked to high molecular weight polylysine is also active. Natural, non-covalent dimers such as the RNase dimer (Park and Raines 2000) often have modulated activity. It is expected that the zero-length cross-linking will also modulate the biological activity and properties of the protein. However, these specific effects will have to be investigated in detail for each protein or combination of proteins that are cross-linked.

Zero-length cross-linking of proteins has rarely been achieved. The in vacuo cross-linking procedure provides a simple and convenient procedure for achieving such cross-linking. The result obtained with RNase A shows that the covalent dimer is formed by only one amide cross-link. However, it has yet to be determined whether a single homogeneous cross-link predominates or whether there are multiple cross-linking sites formed between different carboxylate and amino groups. We believe the former is the most likely possibility; if this is the case, proteolysis of the covalent dimer should yield only one major cross-linked peptide and the latter will yield multiple cross-linked peptides. Further investigations with proteins that have a variety of physical and structural properties are required to determine the generality of the results reported in this communication. The number and location of zero-length cross-links observed in a particular protein and their effects on its activity should provide novel information on structure–function relationships. In other cases, zero-length cross-linking of a protein or proteins may confer advantageous properties that are useful for applications in medicine and industry.

Materials and methods

Bovine pancreatic RNase A (Type I-A), lysozyme, poly-D-lysine, poly-D-glutamic acid, and D(+)-trehalose were purchased from Sigma-Aldrich. All other chemicals, reagents, and solvents were high purity preparations obtained from reliable commercial sources.

In vacuo cross-linking procedure

Lyophilized protein was obtained from the supplier, reconstituted in distilled water to a concentration of 10 mg/mL, and the pH of the solution was adjusted to 7.0 with 1 N NaOH. The protein solution was placed in a glass tube and lyophilized. These glass tubes were sealed under a vacuum of approximately 33 × 10−3 mBar and then placed in an oven at 85°C for 24 h. The vacuum was released and the protein sample reconstituted with 0.2 M Na2HPO4 and 0.15 M NaCl at pH 6.55 to give a final protein concentration of 10 mg/mL.

In some cases, the protein was reconstituted in distilled water (dH2O), instead of buffer, to a concentration of 10 mg/mL, an aliquot was removed, and then the solution was relyophilized and heated again under vacuum at 85°C for an additional 24 h. After four successive cycles of lyophilization, heating, and reconstitution, the final lyophilized protein sample was reconstituted with 0.2 M Na2HPO4 and 0.15 M NaCl at pH 6.55 to give a final protein concentration of 10 mg/mL.

The effect of pH, counter ions, or excipients

Protein solutions (10 mg/mL) at pH values varying from 3.0 to 10.0 were prepared by the addition of 1 N NaOH or 1 N HCl with a micro-syringe, as required. The protein solutions were lyophilized and subjected to the in vacuo cross-linking procedure.

Protein solutions (10 mg/mL) were also prepared in the presence of different cations by the addition of excess LiCl, NaCl, or CsCl followed by dialysis against distilled water. Samples were treated as described above except that the pH was adjusted to 7.0 with 1 N LiOH, 1 N NaOH, or 1 N CsOH, as appropriate.

A solution of RNase A (10 mg/mL at pH 7.0) was lyophilized in the presence of D-trehalose at w/w ratios of protein/trehalose of 5 : 1, 1 : 1, and 1 : 5 and then subjected to the in vacuo cross-linking procedure. On completion, the excess trehalose was removed by dialysis.

Heterogeneous cross-linking in vacuo

A solution containing RNase A and lysozyme in equal amounts (10 mg/mL) was prepared and the pH was adjusted to 7.0 with 1 N NaOH before lyophilization. The in vacuo procedure was performed on the mixture of these two proteins as previously described.

Poly-D-lysine (Mr ~340,000) or poly-D-glutamate (Mr ~32,000) was mixed with RNase A in solution in a 5 : 1 w/w (protein/polymer) ratio. After adjusting the pH to 7.0, the mixture was lyophilized and subjected to the in vacuo cross-linking procedure.

Detection and quantification of cross-linked protein

Cross-linked products were detected by SDS-PAGE with the BioRad Mini-Protean II electrophoresis system. Protein (5–20 μg) was loaded onto a 16.5% Tricine SDS-polyacrylamide gel. After electrophoresis at 130 V for 90 min, the gel was stained with Coomassie Brilliant Blue G250. The relative amount of protein present in each band was determined using the pixel-counting application in ImageQuaNT 5.1 (Molecular Dynamics).

SEC was performed using two Superdex G75 HR 10/30 columns (Amersham-Pharmacia) attached, in tandem, to a Pharmacia fast performance liquid chromatography (FPLC) system with detection at 210 nm. Mobile phase (0.2 M Na2HPO4 and 0.15 M NaCl at pH 6.55 at 4°C) was used at a flow rate of 0.05 mL/min. In general, 0.5 mL fractions were collected. Molecular weight standards (phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; bovine erythrocytes carbonic anhydrase, 29 kDa; trypsin inhibitor, 20.1 kDa; α-lactalbumin, 14.4 kDa) used in column calibration were purchased from Amersham Pharmacia Biotech. Pooled fractions containing RNase A dimer were concentrated to 0.5 mL with Centriprep (Amicon) 3000 molecular weight cut-off concentrators.

RNase A in-gel activity assay

Cross-linked RNase A products were tested for catalytic activity with an RNA agarose gel-based assay (Leland et al. 1998; Gaur et al. 2001). Cross-linked RNase A products (2 ng) were incubated with 5μg of total rat liver RNA in 100 mM Tris-HCl, pH 7.5, containing 10 mM DTT in a total reaction volume of 10 μL. Reaction was allowed to proceed for 10 min at 37°C and was stopped by the addition of 1 μL of diethyl pyrocarbonate and followed by incubation on ice for 2 min. Samples were supplemented with 2 μL of RNA gel loading buffer (10 mM Tris-HCl, pH 7.5, 50 mM EDTA), glycerol (30% v/v), xylene cyanol FF (0.25% w/v), and bromophenol blue (0.25% w/v) before loading onto 1.5% agarose gel containing 2% formaldehyde and 0.05 M ethidium bromide.

Chemical modification of proteins

Dimethylation of amino groups was performed on RNase A (15 mg) according to the procedure described by Means and Feeney (1971). Excess reagent was removed by dialysis.

Amidation of carboxyl groups was performed on a solution of RNase A (15 mg/mL in a total reaction volume of 1 mL) in 1.33 M glycinamide at pH 4.75 with activation by carbodiimide, as described by Means and Feeney (1971). On completion, excess reagent and by-products were removed by dialysis.

Mass spectrometric analysis

The nanospray mass spectrum was obtained with a Micromass Q-Tof mass spectrometer. The mass spectroscopic data was deconvoluted by use of MaxEnt1 software (Micromass Ltd.) to provide the singly charged average masses. The RNase sample was purified by standard ZipTip (Millipore) methodology. Analytes were eluted with 75% methanol, 25% water, and 0.2% formic acid; the sample was centrifuged (6000 rpm for 2 min), and then 2 μL was loaded into a gold coated nanospray needle (New Objectives Picotip). The key variable mass spectroscopic voltages included the following: capillary (+950 V), cone (+47 V), and RF lens 1.05; the source temperature was 80°C, and the data for each scan were collected for 5 sec over the range of 400 to 2500 Da by use of an NaTFA solution for external calibration.


This research was supported in part by a grant from the Natural Science and Engineering Research Council of Canada (Grant No. OGP0090550) and, in part, by Health Canada. The in vacuo cross-linking procedure is the subject of Canadian and U.S. patent applications.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4390102.


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