Size Measurement of the Proteins RNAse A and Proteinase K and the Effects of Protein Binding

Written by: Douglas Mankiewicz, Bruce B. Weiner Ph.D., Yuanming Zhang Ph.D., and Jean-Luc Brousseau Ph.D.

Abstract: Brookhaven Instruments was supplied two protein samples by Northeastern University. The protein size was measured using Brookhaven’s NanoBrook Omni instrument. The proteins were measured individually and then the samples were combined in to one mixture to determine if protein binding occurred.


Northeastern University, through Dr. Meni Wanunu and the Physics department, contacted Brookhaven Instruments with interest in measuring the hydrodynamic radius of their proteins, RNAse A and Proteinase K, utilizing dynamic light scattering. The protein sizes were to be measured individually in a given buffer and then the proteins were to be combined then measured again in an approximate 1:1 mixture. The hypothesis was that the size of the combined proteins would appear to double as the proteins binded to one another.

Material Method

The RNAse A was purchased directly from Thermo Scientific came supplied in a buffer of 50 mM Tris-HCl (pH 7.4) and 50% (v/v) glycerol. The Proteinase K was also purchased directly from Thermo Scientific and was supplied in a buffer of 10 mM Tris-HCl (pH 7.5), containing calcium acetate and 50% (v/v) glycerol. Northeastern University supplied a buffer of 1 M KCl in 20 mM Tris pH 8.1.

Northeastern University supplied Brookhaven Instruments with 0.3 mL aliquots of each protein suspension. Brookhaven diluted the proteins to 1.2 mL using 3x 0.1 μ filtered 1 M KCl in 20 mM Tris pH 8.1.

Hydrodynamic radius measurements were performed using a Brookhaven Instruments NanoBrook Omni which utilizes dynamic light scattering as the sizing technique.

The protein samples were measured in BI-SM50, disposable 50 μL low volume cuvettes. Protein samples when combined were added to a BI-SCP 2.2 mL to 4 mL cuvette.

Measurement data was calculated and the reports generated using Brookhaven’s Particle Solutions software.


Figure 1 shows the hydrodynamic radius for RNAse A. The peak radius measured was 2.65 nm. Figure 2 shows the hydrodynamic radius of Proteinase K. The peak radius was 3.35 nm. These results are somewhat larger than might be expected on the basis of the molecular weight of these globular proteins. However, the buffers supplied contained impurities that are consistent with aggregation.

image of measured peak radius for RNAse A
Figure 1: Measured peak radius for RNAse A at 2.65 nm
image of measured peak radius for Proteinase K
Figure 2: Measured peak radius for Proteinase K at 3.35 nm

Finally Figure 3 shows the peaks that were measured when the two proteins were combined. The new measured effective radius is 4.27 nm. There clearly are two peaks in the measured result, the first peak is measured at 2.26 nm and the second peak is measured at 14.7 nm.

image of mixture of RNAse A and Proteinase K
Figure 3: 1:1 mixture of RNAse A and Proteinsase K. Effective radius is 4.27 nm

It could be surmised from these results that, when mixed, the proteins begin to bind and form a larger molecule. It appears that not all of the RNAse had fully binded which is indicated by the 2.26 nm peak.

Although further experiments with various proteins are necessary to establish the robustness of this volumetric measurement, discrimination of isolated proteins from their RNase & Proteinase K complexes highlights the protein size sensitivity of our measurements. Future experiments may focus on measurement of other proteins, detection of conformational changes in proteins, and the effects of electrolyte strength and pH on the measurements.


1. Larkin et al., High-Bandwidth Protein Analysis Using Solid-State Nanopores, Biophysical Journal (2014),

Applications: BiopharmaEnzymes
Instruments: NanoBrook Series